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WEBCHAT GUIDELINES 1. One question per member plus one follow-up. 2. Keep your question brief. 3. Don't moan if your question doesn't get answered. 4. Do be civil/polite. More here.
Live webchat with Slummy Mummy columnist and author, Fiona Neill(83 Posts)
Fiona Neill is the creator of the Times Magazine Slummy Mummy column and author of international bestseller The Secret Life of a Slummy Mummy which was voted Mumsnet Best Light Read in 2011.
Fiona's latest book, What the Nanny Saw is out on 18th August and to mark the occasion,Fiona will also be joining us at MNHQ for a live webchat on the 18th August, where you'll get chance to tell her your thoughts on the book and to ask her questions.
What the Nanny Saw tells the story of penniless student Ali Sparrow, whose life changes overnight after answering an ad for a nanny. She is catapulted into the privileged and excessive world of London's financial elite, and is a witness to things she probably shouldn't see. When a scandal erupts that rocks the family's private life, is Ali principled enough to keep their secrets when the press come prowling for the inside scoop? Or will she dish the dirt on the family who never saw her as anything other than part of the scenery?
Don't forget to return to this thread to share your views on the book, and put 18th August in your diary to join our chat with Fiona Neill.
thanks for coming along and answering these questions Fiona
I like the fact that you "got" the way a nanny is crucial to a family then discarded when it suits them
I've worked as a nanny and felt this feeling strongly
If you need any more insights from an ex-nanny, I'd be more than happy to help
oh and cheers for the free book, its being passed amongst my friends as we speak!
I only posted my question as felt bad as the chat was rather dead, then I got ignored, lol
Thanks everybody who took part - and thanks Fiona.
Bye everyone. I am a mumset 'lurker' (correct terminology?) thanks for having me www.mumsnet.com/microsites/what-the-nanny-saw if you didn't get a copy of the book there is extract on this link all best fiona
Hello again, sorry a bit late to the party as running around somewhat this morning. Thank you for answering my question - DD2 has flown off this morning taking the book with her to finish it en route to the UK. She just wanted me to say to you 'thank you for the most unputdownable book I have read all summer'. Given that she is not quite 17 and is generally more interested in what the Z listers are up to, I can promise you that this is a huge amount of praise!! She has also removed the Slummy Mummy books from the shelf as she feels they will be far more fun than her prescribed A level reading next term
Hello, thanks so much for your lovely comment. I'm so thrilled your seventeen year old enjoyed the book. If she loses it let me know and i'll get another copy to you. It's great to know it appeals to a wide age range. You don't write with anyone in mind so it's always gratifying to know who enjoys it.
Hello Fiona
congratulations on another well received book. I was lucky enough to receive a copy and am enjoying it all so far (Chapter 3 or so - although it hasn't bitten yet I am intrigued ....)
As I haven't finished this one yet I can't ask too much but With your Slummy Mummy Series - did you feel. once published, that your parenting skills were more open to critisism? (Did you find yourself behaving different in public to show that your wittty asides were just that or you felt like you had to play to the crowd?)
Hello again. I wasn't aware of anyone questioning my parenting skills after slummy mummy although they would probably be too polite to say anything to my face! Most people who know me, realize that Lucy Sweeney and her band of friends are fictional characters. Unfortunately I'm far more anxious about things. I agree with her 'good enough parenting' approach and actually that doesn't really leave you open to too much criticism.
Seeing as it's quiet in here I'll ask another. How was Columbia and was it a gritty research trip or an escape it all and chill?
And did you come across anything to do with (innovative) the Crossed Legs Strike ?
Hello BonzoDooDah. Colombia was great, I've been there quite a lot before because I used to live and work in Latin America. We were visiting my husband's family in Bogota and then went north to the coast to Cartagena. Have had lots of emails from them worrying about how dangerous it is living in the mean streets of London. Will answer your other question below!
I'm afraid I haven't finished it in time for the webchat but I am really enjoying it. I like Ali as a character. Is she based one someone you know in real life? And if she is, do they know?
Hello Blatherskite. Ali is completely fictional. I needed to create a down to earth, intelligent and un-judgemental character who could observe the Skinner family fairly dispassionately. I was brought up in Norfolk and can identify with her need to escape and then the pull back home. There is something about the Norfolk landscape that means you never really leave and I still love swimming in the sea whenever I can. I grew up very near to Cromer, which is a town I still love for its utter authenticity.
<<waves hand feebly from the back>>
Just rushed in, glad I haven't missed the chat >wheezing<. Hi Fiona. Do you use a nanny with your kids? How do you balance work and parenting?
Hi Stranger with my face. When I went back to work after the birth of my first child I did employ a nanny but she didn't turn up the first day I was due back in the office. So I ended up using a nursery and my mother in law. Later I had a German au pair (who lasted six weeks) and then a lovely Slovakian au pair. I now have help in the afternoons during term time and sometimes an eighteen year old student helps me in the holidays. In short it's a patchwork of muddle and juggle. I often get up really early in the morning because I love that feeling of being ahead before the day starts. If I worked in an office full time I'd definitely employ a nanny.
I'm afraid I haven't finished it in time for the webchat but I am really enjoying it. I like Ali as a character. Is she based one someone you know in real life? And if she is, do they know?
I'm looking forward to reading it this hols, especially as I used to be a nanny to two (fairly posh) families, before I had my own two
really enjoyed slummy mummy, love your sense of humour and look forward to reading the new book.
Interested as a writer, how do you find the best approach to capturing your ideas, do you have a set time each day, keep notes etc.
I would love to be able to put pen to paper , not sure where to start though?
It happens in different ways. Sometimes a character comes to you (in this novel Foy existed in my head many years ago after a conversation I overheard on an airplane) and sometimes different scenes write themselves. The kernel of the idea for this novel came from a news story I read in the New York Times. It was about a woman who worked for a financial pr company in New York who discovered that her banker husband was spying on her to do insider trading. I carry notebooks with me all the time and jot down notes. I have lots of ideas on the go at once! When I am ready to write, I am very disciplined because I know that I only have six hours while the children are at school. At the beginning I try to write 1,500 words a day. But I don't beat myself up if I miss the target. I would recommend doing a thorough plot outline and character profiles. That way you can avoid wasting valuable time.
Hello again, sorry a bit late to the party as running around somewhat this morning. Thank you for answering my question - DD2 has flown off this morning taking the book with her to finish it en route to the UK. She just wanted me to say to you 'thank you for the most unputdownable book I have read all summer'. Given that she is not quite 17 and is generally more interested in what the Z listers are up to, I can promise you that this is a huge amount of praise!! She has also removed the Slummy Mummy books from the shelf as she feels they will be far more fun than her prescribed A level reading next term
Just rushed in, glad I haven't missed the chat >wheezing<. Hi Fiona. Do you use a nanny with your kids? How do you balance work and parenting?
I finished it at the weekend and thoroughly enjoyed it. All the loose ends nicely tied up and not in a predictable way either. Would love a sequel as some of the characters were so appealing (and so well written) that I want to know how things turn out for them, especially the adorable little twins. Well done Fiona, ten out of ten from me!
Thanks for your observations Kathleen. Really glad to hear you enjoyed it. I don't have any intention of writing a sequel because when you finish writing a book you want to take a break from the characters you have spent the past couple of years writing. But I do miss Ali and Foy! A friend of mine with identical twins really helped me to understand how they interact and I found it really fascinating. Hope they feel real to mums with twins! There is some interest in creating a four part TV series so I'll keep Mumset posted if that happens.
I have just finished it, I found the beginning layout a little difficult to get into. However, I was soon ploughing through the book. I
t was an easy read and provided good escapism, I also enjoyed how chunky it was a a volume. However, I would have changed the ending slightly if I could as I found the focus was more on the family, when I would have preferred it to be on Ali. Still, a good read and I am so pleased I managed to get a copy as I am not usually so lucky!
Hi purplebuns. Not sure what to say about the beginning, difficult to analyse your own book. But I can say about the ending that I wanted to underline the way that at the end of the day, Ali was someone that the family dispensed with as soon as she ceased to be useful to them. She was an adjunct to the Skinner's life, whereas she felt totally absorbed by their family. I also wanted to look at the way she was living her life through the family and how she fell foul of them as soon as she made the transition from observer to participant. Thanks for your comment.
really enjoyed slummy mummy, love your sense of humour and look forward to reading the new book.
Interested as a writer, how do you find the best approach to capturing your ideas, do you have a set time each day, keep notes etc.
I would love to be able to put pen to paper , not sure where to start though?
Are you a MN regular, Fiona?
Slummy mummy I mean.
I really enjoyed the slummy mummy book. Looking forward to reading your new one.
Was it slightly autobiographical or total fiction?
Received a copy (thanks Penguin!) and enjoying the escapism; wondering if this is really how the other half live.
n.b. Just looked at its entry on a popular online bookshop : I wish books about women's lives today weren't all reduced to being defined as 'chick lit'. Patronising or what?
Hello just a girl from edgeware. I did enough research around the streets of Holland Park and enough interviews to know that this is really how the other half live. (Although this group of people is much less than a half, a tiny but significant and powerful minority perhaps). Agree totally with the chick lit label. It's so reductive and encompasses so many female writers that it isn't really helpful because it doesn't describe the range of writing. Also it's inevitably a pejorative term. but people love labels and it probably isn't going to disappear.
Could hardly put it down. Very good characterisation in the book, plausible scenarios, shrewd insights, and I found it good to read as a companion piece to Kathryn Stockett's 'The Help'. Great job.
My question. I thought it was particularly interesting when you wrote about the way the bankers' families apparently commoditised everything, including relationships. I was wondering whether you had experienced seeing this sort of thing up close in real life and whether writing the book was a way of being able to make comments about certain behaviours that would be socially unacceptable face to face (I ask because I certainly have seen this sort of thing at first hand and it turns my stomach, but obviously it's difficult to say anything while you are there).
Thanks so much Senior Wrangler. I'm really glad you enjoyed it. It took a lot of research and a lot of time. I interviewed a lot of bankers to write the book, as well as nannies who had worked for super wealthy families. The bankers mostly helped me out on the finer points of derivatives trading but the nannies were a rich resource in terms of the often hilarious accounts they gave of life in fully staffed households in central London. I think that there are now quite a lot of very wealthy families based in London, who have the financial capacity to buy their way out of many of the more unpalatable aspects of parenting and domestic life.
Thanks for answering my question and I'm sorry if my comment about the opening chapter sounded rather rude- I cetainly wouldn't have said it to your face!
The character of Foy certainly rang true!
Hi Fiona
Is the relationship between nanny and mother pretty much designed to be tortured? Apart from the odd exception, I don't know anyone on either side who finds it particularly easy..
Hello there. I tried to depict the awkwardness of the relationship but tried to avoid making it too torturous (I hope). It can sometimes be an awkward relationship, particularly I think if the nanny lives in with a family. Interviewing nannies before writing the book I was struck by the difficulties inherent in being at the heart of the family one moment and then expected to slip back into the shadows the next. It is a psychologically complex role.
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Tesla adds James Murdoch and Linda Johnson Rice to its board
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Tesla has added two new independent directors to its board, including 21st Century Fox CEO James Murdoch and Johnson PublishingRead more
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Cutoffs and k-mers: implications from a transcriptome study in allopolyploid plants
Transcriptome analysis is increasingly being used to study the evolutionary origins and ecology of non-model plants. One issue for both transcriptome assembly and differential gene expression analyses is the common occurrence in plants of hybridisation and whole genome duplication (WGD) and hybridization resulting in allopolyploidy. The divergence of duplicated genes following WGD creates near identical homeologues that can be problematic for
Here we report a successful strategy for the assembly of two transcriptomes made using 75 bp Illumina reads from
To obtain optimal transcriptome assemblies for allopolyploid plants, k-mer size and k-mer coverage need to be considered simultaneously across a broad parameter space. This is important for assembling a maximum number of full length ESTs and for avoiding chimeric assemblies of homeologous and paralogous gene copies.
Whole genome duplication (WGD) associated with autopolyploidy and allopolyploidy has been a recurrent and prevalent phenomenon in plant evolution, linked to species diversification and species radiation
Transcriptome assembly has "many informatics challenges"
Several pipelines for the assembly of transcriptomes have been suggested in a number of studies. These studies have identified k-mer size as an important parameter. During contig assembly assemblers such as ABySS, Trans-ABySS and Trinity, k-mer size specifies the length of an oligonucleotide sequence used for building the
Here we report a successful strategy for the assembly of two reference transcriptomes in
Quality assessment of the reads and
Two lanes of paired-end and one lane of single-end Illumina 75 base pair sequences were generated for
Assessing the assemblies
For each of the 380 assemblies the number and length of the contigs was assessed. In total 23,668,704 contigs were assembled for
Additional file 1
Table S1. Number and length of contigs per k-mer size and coverage cutoff for
Click here for file
We also compared the total number of assembled bases for each assembly. The highest number of assembled bases for
In order to determine the percentage of reads included in each assembly we mapped the reads of each species against the respective contigs of each assembly. In
For each combination of assembly parameter values the length of the longest sequence was determined and annotated against homologues in
Additional file 2
Table S2. Statistics and longest sequences for 380 assemblies of the
Click here for file
Additionally, the N50 and N90 lengths were computed for each assembly of
The importance of the k-mer size and the coverage cutoff for transcript assembly
Figure 1
Number of complete transcripts identified in different assemblies of
Number of complete transcripts identified in different assemblies of
Additional file 3
Figure S1. Number of complete transcripts identified in different assemblies of
Click here for file
Additional file 4
Supplementary file S1: 3,912 complete annotated transcripts from the library of
Click here for file
Additional file 5
Supplementary file S2: 2,442 complete annotated transcripts from the library of
Click here for file
Figure 2
Number of genes assembled with a certain number of k-mer sizes and coverage cutoffs
Number of genes assembled with a certain number of k-mer sizes and coverage cutoffs. a) For each of the 3,912 complete transcripts in the
Comparing assemblies in terms of the number of complete transcripts
To quantify the similarity of assemblies made using different parameter values we counted the number of complete transcripts in each assembly and made pair wise comparisons of assemblies. For each comparison we divided the number of complete transcripts common to both assemblies by the total number of complete transcripts summed across both assemblies. The highest value therefore was 0.5 for perfect overlap and the lowest value was 0 if no sequence was identical between the complete sequences of the two assemblies. These values were then divided by 0.5 to regain easily comparable percentages (see Figure
Figure 3
Amount of overlap between complete transcripts identified in different assemblies of
Amount of overlap between complete transcripts identified in different assemblies of
Comparison to trinity assembly
Most similar homologues
All ABySS contigs of
Sequences of
The lengths of the contigs with hits in the plant database were determined as well as the lengths of the contigs without those hits. Both length distributions were then compared using a Wilcoxon rank sum test. The length of the contigs with hits was significantly longer than the ones for the other sequence set (p < 2.2e-16). The mean length of the contigs with hits was 252 while it was 199 for the other sequence set. The longest sequence in the first set is 10,134 bp while the longest sequence in the other set was 8,292. For both datasets most sequences were between 100 and 300 bp long (77.8%, 86.8%) but the percentage of sequences longer than 1000 bp was slightly higher in the dataset with hits in the plant database (2.2% vs 0.8%). The 776 sequences longer than 3000 bp without hits in the plant database were analyzed further as it is highly unlikely that sequences of this length are comprised of 'nonsense' assemblies. These sequences were found in all assemblies with a k-mer size smaller that 59. When compared against the nucleotide database at NCBI they either hit hypothetical or uncharacterized proteins and genomic sequences. The sequence identity of these hits was mostly below 70%. The longest sequence (8,292 bp; coverage cutoff: 8; k-mer size: 43) did have a hit in the plant database (AT5G40450; uncharacterized protein) but a large number of indels in the alignment reduced the identity to 53%. Interestingly, this sequence passed the filters when searched against the coding sequences of
A comparison of orthologues, paralogues and homeologues
We used two reference transcriptomes for the identification and annotation of homologous transcripts within and between our
We counted the number of homeologous pairs present in both species. 547 homeologous pairs were identified as common to both. The mean sequence identity of these homeologous copies was 98.66%, while the minimum was 95.05%. We used this observed distribution of identities to determine the relationship among other homologues occurring in the two libraries. We inferred sequences to be putative orthologues (that is, copy one in both species) if they had greater than 95% sequence identity, and inferred sequences to be likely paralogues (that is, copy one in one species and copy two in the other) if they had less than 95% identity (see Figure
Figure 4
Schematic of homologous sequences within and between
Schematic of homologous sequences within and between
In order to determine if the 3,206 genes only found in the library of
The sequences present in both libraries (547 homeologous pairs, 4,590 paralogues) were compared to homologues from
Observations on specific genes that highlight issues for assembly
We investigated the number of reads mapping to seven genes that were expressed at different levels and exhibited different degrees of sequence similarity. This was done allowing for no mismatches and also up to 3 mismatches per read. The motivation for allowing mismatches was to accommodate potential sequencing errors that might occur with high density of reads and also to demonstrate the assembly problem caused by having very similar homeologous sequences in the dataset.
Two genes studied (rbcS, ESM1) had a very high expression level. For these a complete transcript was assembled under very few k-mer size and coverage cutoff combinations. Homeologous copies were not assembled for either gene. In the
Figure 5
Assembly patterns in the
Assembly patterns in the
The number of reads mapping to each of these sequences established different expression levels of the corresponding genes. 215,536 reads mapped to the sequence of rbcS and 195,295 to ESM1, only 10,937 reads mapped to one homeologous copy of MVP1, while 1,854 reads mapped to the other homeologous copy. 8,903 reads mapped to the paralogue of MVP1. 3,579 reads mapped to rbcL and 3,420 to the homologue of AT1G75680. When allowing for up to three mismatches, the number of additional reads mapping to the sequences did not scale proportionally. While the number of reads mapping to ESM1, rbcL and the homologue to AT1G75680 increased by about 9%, it increased by 28% for rbcS and 229% for one copy of MVP1. The number of reads mapping to the other two sequences of MVP1 increased by 10% and 14% (see Table
Table 1
Number of reads mapping to seven example transcripts.
sequences/# mismatch
# assemblies
The number of reads from the
A comparison of assembled MVPI homeologues in the
Separate and joint assembly of reads for specific genes
Reads mapping to assembled ESTs for the seven mentioned genes were removed from the pool of reads. Reads mapping to each of these genes were then assembled separately with k-mer lengths 25 to 63, and without specifying a coverage cutoff. When no coverage cutoff is specified, ABySS determines the coverage cutoff automatically according to the actual k-mer coverage of each respective sequence. The automatically determined coverage cutoffs for individual genes varied greatly. For example, while the determined coverage cutoff for rbcS was between 46.8 for k-mer size 63 and 122.8 for k-mer size 25, it was between 4 and 9.64 for rbcL (see Additional file
Additional file 6
Table S3. Coverage cutoff values for seven genes made from assemblies of reads which had up to three mismatches. All reads in the
Click here for file
To determine the effect of including reads in an assembly where there are mismatches to the contig sequence, the reads mapping to each sequence were assembled with k-mer sizes 25 to 63. Four different read datasets were used for each gene sequence: no mismatch, up to one mismatch, up to two mismatches, and up to three mismatches. If one contig were assembled with each k-mer value (2-63) and for 0-3 mismatches, then 20x4 (80) full length identical transcripts are expected for each gene. However, the resulting contigs not only varied in length and number but also in their coverage. After the separate assembly of the seven example genes, we combined the datasets of all seven genes to simulate a small transcriptome assembly.
In the separate assemblies, the genes with high expression levels (ESM1, rbcS) were assembled to a full length transcript in most assemblies. The assembly of ESM1 was the least sensitive to changes in parameter values. With this gene, there was no fragmentation as all 80 assemblies each produced exactly one contig. This was also true for the assemblies of rbcS when the read dataset without mismatches was used. As soon as reads with one to three mismatches were included, some assemblies were fragmented. Full length transcripts were only found with k-mer sizes 61 and 63. The highest fragmentation (eight contigs) was found with k-mer size 27.
In the joint assemblies the results differed dramatically from the separate assemblies. While for ESM1 there was a full length contig for each k-mer when the reads mapping without mismatches were used, 12,930 contigs were assembled when one mismatch was allowed, 15,500 with two mismatches and 16,899 with three mismatches. Most of these sequences were smaller than 120 bp. Some longer contigs (up to 1,103 bp) were obtained in the dataset with one mismatch, though none of those were full length transcripts. The same observation was made for rbcS. While there were 20 contigs for the dataset without mismatches, there were 11,361, 13,636, and 14,287 for the other datasets. No full length transcripts were found for these, with the maximum length transcript being 233.
With the three MVP1 homologues (two homeologues, one paralogue) and the separate assemblies highest fragmentation occurred with small and large k-mer sizes. K-mer sizes between 25 and 51 produced full length contigs for one homeologous copy (except for the first six nucleotides). Increasing the number of mismatches increased the amount of fragmentation and decreased the number of full transcripts obtained. The assembly of the duplicated MVP1 gene sequence showed the least fragmentation of MVP1 homologues.
The results for the joint assemblies were similar. For the first homeologous copy of MVP1 no full length transcript was found. Full length transcripts were found for the second homeologous copy with k-mer sizes 49 to 59 in the dataset without mismatches and 51 to 61 in the others. The lowest degree of fragmentation again was found for the third sequence of MVP1. Full length transcripts were assembled with k-mer sizes 29 to 55. In the other assemblies, the sequence was fragmented into three contigs.
The lowest degree of fragmentation in the separate as well as the joint assemblies was found for the two sequences with a low expression level. Only assemblies with large k-mer sizes, regardless of separate or joint analyses, failed to produce fully assembled sequences. Allowing for mismatches where genes had low expression levels resulted in a decrease of fragmentation in assemblies with high k-mer sizes indicating that the additional reads were crucial for the assembly of regions with low coverage.
Gene expression levels and assembly parameters
In order to determine whether or not a relationship existed between the expression level of a gene and assembly parameters as previously suggested
In this study we investigated the difficulty of transcriptome assembly in the case of an allopolyploid transcriptome in which there were high levels of similarity between homeologues. Our findings supported earlier studies which have identified the importance of k-mer size for optimal assembly
Comparison of homologues
The parental species of the genus
This high degree of similarity between homeologues created a high risk of assembling chimeric sequences, where one part of the sequence derives from one copy while another part derives from the other copy. Furthermore we wished to avoid assigning contigs for different homeologous copies to the wrong copy. For this reason we only evaluated contigs that were assembled to be longer than 55% of the reference gene to which they were annotated. This minimal length ensured a minimum of 5% overlap between
Interestingly, only 35% (5,684) of the genes that were unambiguously identified were present in both libraries. Among these, both copies were present for 547 genes, while for 4,590 genes only one copy was identified in both species. For 65% (7,600 and 3,206) of the assembled sequences no counterpart was found in the respective other library although for a surprisingly high number of genes (1,122) the respective second copy was found in either one of the two libraries. This relatively small percentage of overlap between the assembled libraries and greater number of sequences in the
The comparison of homeologous pairs found in both species with their homologues in
The mean length of the sequences without a hit in the nr plant database is slightly smaller than the length of sequences with a hit, confirming the observation that the annotation rate for sequences shorter than 200 bp is not as reliable as for longer sequences
K-mer size and assembly
Comparison of assemblies made with different parameter combinations showed that the k-mer size had a significant impact on the length of contigs as previously observed
Coverage cutoff and assembly
Although it appears not to have been used to advantage in reported transcriptome studies, the coverage cutoff can also help to avoid assembly problems when there are identical regions between homeologues or paralogues. If one of two highly similar homeologues has a high expression level while the other homeologue has a low expression level, the highly expressed homeologue can be assembled using high coverage cutoffs. Doing this will exclude from assembly the low coverage reads that belong to the second homeologue. Once the highly expressed reads have been assembled (and removed from the pool of reads) then the assembly of reads from the lowly expressed homeologue can be made using a low coverage cutoff and a small k-mer size. Indeed it may make little sense to search for a single set of 'best' assembly parameters for a transcriptome. Such an approach is likely to limit the number of genes that can be assembled in the EST library.
Trans-ABySS and Trinity
The Trans-ABySS
Assessment of assemblies
Parameter estimates used to assess
Assessment should be based on the total number of full length transcripts
While it is obvious that there must be one best assembly with regard to whole genomes
In our study, the highest number of full length transcripts was found with k-mer size 41 and coverage cutoff seven for
Comparison of assemblies revealed a surprising lack of overlap with respect to the full length transcripts. The maximum number of full length transcripts found in one assembly was 741. If only this assembly had been conducted, 3,171 sequences would not have been assembled to full length transcripts. For many genes near identical parameter values gave similar assembly results (60-80% overlap), while more distinct parameter combinations produced assemblies with little overlap. Transcripts found to be full length under one set of assembly conditions often occurred in other assemblies in a more or less fragmented state. Such fragmented sequences are less useful for differential expression analyses as the statistical power is less for smaller sequences
The realization that an optimal assembly requires optimization for each gene becomes even clearer when the parameter combinations for which full transcripts were assembled are considered. There was no gene in our dataset whose assembly was not influenced by either the coverage cutoff or the k-mer size. For example, although there were some genes in
A higher expression level typically is associated with a wider range of optimal assembly parameters (Figure
The three gene copies for MVP1 are highly similar and thus require assembly using higher k-mer values. However the transcripts for these copies have a low to medium expression level, which means that high k-mer values are not appropriate. A tradeoff seems to be k-mer sizes 51 and 53 with which all sequences can be assembled to nearly full length transcripts.
Assembly of the transcripts for rbcL and AT1G75680 required accommodating low levels of gene expression. In this situation contigs might not be joined because there are too few reads connecting them. Including reads with mismatches in this instance is expected to help the assembly as the presence of these can increase read coverage. This was found to be the case in the assembly of rbcL. This gene is chloroplast encoded, and therefore only one copy of this gene exists, thus there were no reads stemming from a similar homeologous or paralogous copy to interfere with the assembly. This also seems to have been the case with AT1G75680. That is, while it is nuclear located, the inclusion of mismatched reads improved the assembly regardless of the k-mer size and cutoff values. This hints either towards the presence of only a very dissimilar homeologous copy in the transcriptome or towards a lack of expression of a homeologous copy.
In summary, our observations suggest that common causes of fragmented assemblies with allopolyploid libraries are either too low or too high expression levels and a high degree of similarity between homeologous or paralogous sequences. All of these issues can be addressed using different strategies, however the success of these strategies depends on multiple features of the genes. For example, the inclusion of mismatched reads can assist in the assembly of transcripts with a low expression level. However, the addition of mismatched reads can introduce significant noise into a dataset of reads when transcripts are highly expressed. In this instance their inclusion will be unhelpful.
As many gene families have arisen through gene duplication during evolution the similarity between different gene copies can pose a problem of significance in the assembly of transcriptomes. This is especially the case for organisms of polyploidy origin. The problem for assembly that highly similar gene copies cause can to some degree be overcome by altering the k-mer size and the coverage cutoff as shown here. While the addition of longer reads into the assemblies can also be expected to provide a solution for these cases, our findings highlight the potential of using even very short reads for the assembly of allopolyploid plant transcriptomes.
Plant growth
For the paired-end sequencing two
For single-end sequencing, seeds of
RNA extraction and sequencing
Total RNA for each sample was extracted using the RNeasy plant mini kit (QIAGEN, Hilden, Germany).
For the paired-end sequencing, RNA from young and old leaves was pooled in equal amounts before sample preparation for mRNA sequencing. Next, the combined leaf RNA and the root RNA from both
For the single-end sequencing, the three replicate
Quality assessment
The quality for each of the Illumina GAIIx 75 bp reads in all eight datasets from
The reads for each species were assembled using ABySS v. 1.2.5
For both species, the contigs from all 380 assemblies each were first analyzed separately. The number of contigs for each k-mer and coverage cutoff was determined as well as the number of sequences that were longer than 1000, between 500 and 1000, between 200 and 500, and between 100 and 200 nucleotides (for details see Additional file
All contigs that were identified as homologues to the same reference sequence (or its homologue in the respective other reference species) and covered more than 55% of that sequence were pooled together and further assembled using the overlap assembler CAP3
Assessment of the assemblies
The sequences of both libraries were compared to the sequences of the same library to identify potential homeologous sequences as well as to the respective other library in order to identify orthologues using BLAST
Those contigs that spanned at least 95% of a reference sequence were extracted from the assemblies. BLAST was then used to determine the amount of overlap between the complete transcripts of two assemblies using an identity cutoff of 100%. The number of identical sequences between these datasets was determined using a Perl script and divided by the sum of number of complete transcripts in both datasets. A perfect overlap of two datasets resulted in a value of 0.5. These values were then divided by 0.5 to regain easily comparable percent values.
Gene expression levels
The expression level of the completely assembled genes was derived by mapping all reads to the sequences of these genes and normalizing this value using following formula for each gene X
where MX is the number of reads that mapped to the sequence of gene X, MA is the number of all reads mapping to all sequences, and LX is the length in kilobases of the sequence of gene X.
All statistical tests were conducted using R version 2.12.2
Identification of best BLAST hits
The sequences that were longer than 100 bp of all 380 assemblies of
BLAST: Basic local alignment search tool; NCBI: National center for biotechnology information; Nr: Non-redundant protein databank; TAIR: The
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
NG assembled the data, analyzed the assemblies, designed the figures and drafted the manuscript. CV provided the plant material, extracted the RNA, and participated in drafting the manuscript. OD participated in the assembly of the data, and participated in drafting the manuscript. PL participated in the design of the study, and in drafting the manuscript. MB participated in drafting the manuscript, and provided the plant material. CE conducted the BLAST searches for the contigs of
CV acknowledges support from the AvH foundation and the Allan Wilson Centre for Molecular Ecology and Evolution. NG and OD were supported by a fellowship within the Postdoc-Programme of the German Academic Exchange Service (DAAD). PL's contribution to this study was funded through a James Cook Fellowship from the Royal Society of New Zealand.
Widespread genome duplications throughout the history of flowering plants
Ancestral polyploidy in seed plants and angiosperms
The flowering world: a tale of duplications
Speciation genetics: current status and evolving approaches
Next-generation transcriptome assembly
Uncovering the complexity of transcriptomes with RNA-Seq
Annotating genomes with massive-scale RNA sequencing
Applications of next generation sequencing in molecular ecology of non-model organisms
Ab initio construction of a eukaryotic transcriptome by massively parallel mRNA sequencing
How to apply de Bruijn graphs to genome assembly
Assembly algorithms for next-generation sequencing data
Short read Illumina data for the de novo assembly of a non-model snail species transcriptome (
De novo assembly and characterization of root transcriptome using Illumina paired-end sequencing and development of cSSR markers in sweetpotato (
RNA-Seq analysis and
De novo transcriptome sequencing in
Optimization of de novo transcriptome assembly from next-generation sequencing data
Expression profiling without genome sequence information in a non-model species, pandalid shrimp (
A Pleistocene inter-tribal allopolyploidization event precedes the species radiation of
A spatial dissection of the
ABySS: A parallel assemble for short read sequence data
Full-length transcriptome assembly from RNA-Seq data without a reference genome
De novo assembly and analysis of RNA-seq data
Transcriptional profiling of an Fd-GOGAT1/GLU1 mutant in
Mapping and quantifying mammalian transcriptomes by RNA-Seq
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs
CAP3: A DNA sequence assembly program
Ultrafast and memory-efficient alignment of short DNA sequences to the human genome
A gene duplication/loss event in the ribulose-1,5- bisphosphate-carboxylase/oxygenase (rubisco) small subunit gene family among accessions of
Modified vacuole phenotype1 is an
Structural organization and a standardized nomenclature for plant endo-1,4-beta-glucanases (cellulases) of glycosyl hydrolase family 9
Velvet: algorithms for
The gene controlling the quantitative trait locus EPITHIOSPECIFIER MODIFIER1 alters glucosinolate hydrolysis and insect resistance in
Identification of non-specific lipid transfer protein-1 as a calmodulin-binding protein in
Stability of AtVSP in the insect digestive canal determines its defensive capability
A multifaceted study of stigma/style cysteine-rich adhesin (SCA)-like
Plant defensins
Probing the structure of Lhca3 by mutation analysis
Analysis of phylogenetic relationships of
High-throughput functional annotation and data mining with the Blast2GO suite
Benchmarking next-generation transcriptome sequencing for functional and evolutionary genomics
BLAT -- the BLAST-like alignment tool
Transcript length bias in RNA-seq data confounds systems biology
SolexaQA: At-a-glance quality assessment of Illumina second-generation sequencing data


text:
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-- Written by a member of TheStreet Ratings Staff
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The dish: Lobster Tacos: Grilled flour tortilla, avocado, diced tomato, scallion, chipotle aïoli and lime, $15
The wine: 2009 Domaine Belluard Vin de Savoie Ayse Brut, Haute-Savoie, France, $14 per glass, $54 per bottle
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The pairing: I love the pairing of Champagne and lobster. This sparkling wine is made in the same painstaking process as Champagne bu
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text: Travel photography dos and don'ts Michael Newington Gray reveals how to take holiday photos that won't bore everyone to tears 27th January 2015 Michael Newington Gray If, after death and taxes, there's a third certainty in life in the 21st century, it's other people's smug holiday snaps.With the rise of social networks and digital photography, everyone can be a travel photographer these days. Which basically means that Facebook, Flickr and Instagram have become the online equivalent of your next door neighbour's post-holiday slideshow hell.I am here to help you save yourself and your friends from this sad situation, using one of the world's most photographed locations, Venice, to demonstrate. While it is both beautiful and unique, Venice has been photographed to a watery pulp by everyone who's ever stepped foot canal-side.With a bit of input from Polish photographer Adam Panczuk (a visiting lecturer at the Istituto Europeo di Design in Venice, in a link-up with the gorgeous Bauers Hotel), here are my travel photography dos and don'ts…DON'T follow the crowd. If everyone's already snapping it, try something else. Do we really need to see another famed world landmark surrounded by day-glo Puffa-jacketed tourists waving their iPhones in the air? No. We don't. Michael Newington Gray Some people taking photographs of each other's backs. DO create something personal, with a bit of atmosphere. How did the landmark feel when you first saw it? Did it take your breath away? Be patient, stand back, wait for that couple with the selfie stick to move out of the way, or try a different angle altogether. Michael Newington Gray Guaranteed no Puffa jackets in sight DON'T take the same photos over and over and over again. Do you expect people to be all, 'Oh my, that bridge is cute! Oh look, another bridge! Is that a floating street? No wait, it's a bridge. Ah, now I see how people get over the water there. HEY LOOK A BRIDGE!'? Michael Newington Gray Like a megaton of bridges of troubled water DO edit! A short set of 5-10 photos can tell a much better story than 50. What really interests you about where you are? The architecture? The history? The light? Try to tell a small story with your images by focusing in on one area or element of a city, and then photographing a series of images. Keep things simple and try to draw out visually striking elements of your surroundings, and think about how you can compose it in an artistic way through the viewfinder. Michael Newington Gray Use trips to tell short storiesSuch as this one to Seguso glass making factory Michael Newington Gray Michael Newington Gray Michael Newington Gray DON'T hesitate. You can always ask later. If you see someone doing something interesting and you don't think they would be offended, take the photo. You want the photo to be natural but you don't want to be chased by an angry mob, either. Michael Newington Gray Although maybe hesitating and getting out of the way of a man with a load of molten glass isn't such a bad idea. DO take photos at different points of the day. The Rialto Bridge isn't going anywhere, but the tourists are. If you're staying near somewhere amazing, try photographing very early in the morning (tricky, I know) or even better, late at night. Turn off the flash and capture your images during the 'magic hour' (when the sun is nearing the horizon). Wherever you are, it will give an amazing natural warmth to your images which no Instagram filter can even hope to replicate. Slithers of light upon the cobbles or warm light radiating from a café can make a mysterious night scene. Michael Newington Gray In the middle of the afternoon, these tourists won't be going anywhere Michael Newington Gray But an early rise could get you a clear shot of the area's most famous landmarks Michael Newington Gray ...Or indeed at night. DON'T use a selfie stick. No. NO NO NO NO NO NO NO. True story: you'll look like a dick, and everyone will hate you. Get your friend/lover/child/attractive tourist in the vicinity to take a picture for you. The end. Michael Newington Gray No one cares no one cares no one cares no one cares no one cares no one cares Francesca Newington Better! The Bauers hotel in Venice has teamed up with the Istituto Europeo di Design to offer a series of workshops with some of the continent's most learned snappers. For more details click hereTickets are also available for the Venice Carnival at the Bauers See also... The Tatler Wolf Hall makeover Miranda Hart's superyacht adventure Emma Freud's gadgets


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The Takeaway
'It's Not the Addiction that Kills People, It's the Smoke'
Each May 31st, the World Health Organization declares another "World No Tobacco Day" meant to encourage a full day of abstinence from all forms of tobacco consumption. But this year, some public health officials are wondering if it's time to change our approach to healthy living. While no one doubts the addictive properties of tobacco, and the negative consequences for all of our health — is it time to stop demonizing an addiction that affects 20 percent of Americans, and think of ways for smokers to give up cigarettes but still have access to nicotine?
Michael Siegel of Boston University says its time to consider new options like the e-cigarette, which delivers a safer form of nicotine.


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DILLARD HOSTS DAY OF ACTION: Youth-led groups will host a Day of Action to End Police Violence at Dillard University from 4 p.m. to 8 p.m. Thursday, Jan. 15 at the Georges Auditorium on the campus of Dillard University, 2601 Gentilly Blvd., New Orleans.
With the support of American Friends Service Committee and Peace by Piece, local youth activists host #DOA — A Day of Action.
The Day of Action is a free event that begins with a live broadcast hosted by TheRealNews.com at 4:30 p.m. of a national discussion with activists from around the country. Following the broadcast is an audience discussion at 5:30 p.m. and a public forum and panel at 6 p.m. Refreshments will be served throughout the evening.
Panelists include Norris Henderson, Voice of the Ex-Offender; Ursula Price, Independent Police Monitor's Office; Devin Sordelet, Dillard University student; and Tracie Washington, Dillard University and Louisiana Justice Institute. The panel discussion will be moderated by RCA recording artist Dee-1.
Topics will include police accountability to the community; challenging stop-and-frisk policies; and encouraging more positive interaction between police and the community.
American Friends Service Committee is a Quaker organization that promotes lasting peace with justice as a practical expression of faith in action. Peace by Piece is a nonviolence youth program that engages young people to be peace leaders in their schools and communities.
For information about the national Day of Action, call (504) 565-3596, or visit soar.afsc.org or facebook.com/peacebypiecenola.
TULANE HILLEL'S BIG ISSUE: Tulane Hillel's next Big Issue panel discussion, will be held Thursday, Jan. 15 at Tulane Hillel, 912 Broadway. Doors open at 7 p.m. and the panel begins at 7:30 p.m. The topic will be "Inns and Outs: Is there room in New Orleans for Airbnb and short-term rentals?"
The panel will be moderated by City Business Editor Greg LaRose and will explore the diverse angles that supporters and opponents of Airbnb and other vacation rental sites, have cited as reasons for their stances.
Panalists include Polly Hardi, president of the Alliance for Neighborhood Prosperity; Boonie Rabe, innkeeper at Grand Victorian Bed and Breakfast and president of the Professional Innkeepers Association of New Orleans; Lisa Suarez, president of the Faubourg Marigny Improvement Association; Rafe Rabalais, project manager of Gulf Coast Housing Partnership; and Mavis early, executive director of the Greater New Orleans Hotel and Lodging Association.
DANNY GLOVER IS KEYNOTE: Efforts to improve employment opportunities for black people will be highlighted during "#BlackJobsMatter: The People's Summit on the Black Jobs Crisis" on Monday, Jan. 19 in the gymnasium at Southern University New Orleans, 6400 Press Drive.
The summit will include a keynote address by Danny Glover. According to a statement, the black jobs crisis in New Orleans includes 52 percent unemployment among black men.
Expert panels and breakout sessions, a career opportunity and resource fair; and cultural expressions on the black jobs crisis.
The event is being organized by the New Orleans Workers' Center for Racial Justice, Stand with Dignity, and the Community Evaluation Commission.
For information, visit nowcrj.org.


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| Home - Latest News | Introduction | Bayside Prophecies | Directives from Heaven | Order Form | Testimonies | Veronica Lueken | Miraculous Photos | Bible | Radio Program |
What's wrong with the Masons?
"My children, and I ask My pastors to listen to Me as your Mother, as I tell you that you must take yourselves from all secret societies. They do not openly plot against My Son's Church, but they do this in secret! The Masons, My children - the Hierarchy must remove themselves from this abominable organization. You cannot deny that many in the Masons are practicing witchcraft and sorcery." - Our Lady of the Roses, June 1, 1978
(The following is taken from the booklet, "Why the Catholic Church says 'Investigate'", + Imprimatur Joseph E. Ritter, Archbishop of St. Louis, January 24, 1949):
Many non-Catholics, whether they be Masons or not, are honestly puzzled by the attitude of the Catholic Church toward Freemasonry. They comment about it along the following lines:
"I have lived among Masons all my life... some of the finest men I have known have been Masons ... they conduct many charitable enterprises and are moral, law-abiding citizens ... what has the Catholic Church got against Masons?"
The answer can be briefly stated, Catholics have nothing against individuals who are Masons, or against the good works carried on under Masonic auspices. But no Catholic can subscribe to Masonic religious teachings or practices, or take the Masonic oath ... much less promote by his dues and active cooperation, official Masonry's hostility to the Catholic Church.
It is not merely because it is a "secret society", that Catholics cannot be Masons. It is because of the particular kind of secret society that Masonry happens to be.
Masonry has a distinctly religious character, a fact which may surprise many American Masons who have joined the Lodge for business, social or political reasons. They will be tempted to deny its religious character. The look upon Masonry as a purely fraternal organization and do not consider it a religion. Either they do not know the true constitution of their Lodge, or Masonic authorities are violating the principles of Masonry, for they definitely emphasize its religious character.
It may be fairly assumed that the Grand Lodge of the State of New York knew what it was talking about when arguing its case against a certain Robert Kopp in a trial before the Appellate division of the New York Supreme Court. Kopp had been expelled from the fraternity and had appealed the action of his brethren to the civil court. One of the strong points made by the Grand Lodge during the trial was as follows: "The right to membership in the masonic fraternity is very much like membership in the church. Each requires a candidate for admission to subscribe to certain articles of religious belief as an essential prerequisite for membership. Each requires a member to conduct himself thereafter in accordance with certain religious principles. Each requires its members to adhere to certain doctrines of belief and action..."
"The masonic fraternity may, therefore, be quite properly regarded as a religious society, shall have sole and exclusive jurisdiction to determine matters of membership, should be deemed applicable to the masonic fraternity." Such a statement speaks for itself.
A brief description of the "articles of religious belief which are an essential prerequisite to membership," can be found in the writings of the Grand Master, Albert Pike, generally admitted to be the best authority on Masonic matters in America. "Masonry propagates no creed," he wrote in 'The Inner Sanctuary,' "except its own most simple and most sublime one taught by Nature and Reason. There has never been a false religion in the world. The permanent one universal revelation is written in visible Nature and explained by Reason and is completed by the wise analogies of faith. There is one true religion, one dogma, one legitimate belief."
Whatever we may say about these principles, certainly no Catholic could subscribe to them, for they flatly contradict his own religious belief. In fact, it is difficult to see how any person who believes in the truth of Christianity can subscribe to them. How can he believe that there is no such thing as a false religion... that Christianity is merely one of many expressions of his universal true religion and that religion is taught by Freemasonry? No doubt, most Masons in America do not believe this, but nevertheless, it is the teaching of Masonry.
"Intellectual Science"
The fact that many Masons are unaware of the religious teachings of Masonry is not surprising. Even Masonic authorities admit that the great majority of Masons are in the dark. "The Masonry of the higher degrees," says Pike, "teaches the great truths of intellectual science; but to these, even as to the rudiments of first principles, Blue Masonry is absolutely dumb." And Oliver wrote: "Brethren, high in rank and office, are often unacquainted with the elementary principles of the science..." Masons "may be fifty years Masters of the Chair and yet not learn the secret of the brotherhood..." In view of the fact that the secrets of Masonry are unknown to the bulk of Masons, the oaths of secrecy taken by Masons are unjustifiable from the Catholic point of view.
It is a serious thing for a person to take an oath without regard to consequences, to oblige himself to actions of which he is ignorant and which may be contrary to the dictates of his conscience. It is morally wrong for a person to take such an oath. No Catholic could swear on the Bible, using the following words, as recorded by Pike: "I pledge myself to obey without hesitation any order, whatever it may be, of the regular Superiors in the Order."
It may be true that many Masons do not look upon their obligations in this light. But this does not make the oath itself any less wrong. Perhaps they would violate their conscience because of their oath but, in so doing, they would not be living up to the official principles of Freemasonry.
"Crush Catholicism"
There are many individual Masons who have nothing but good to say about the Catholic Church and, if anything, they admire it. But they do so as our fair-minded fellow citizens and not as Masons. And in so doing, they are not reflecting the official attitude of Freemasonry which time and time again has expressed its absolute opposition to the Catholic Church.
As late at 1913, the Grand Orient in France issued this statement: "The aim of the Grand Orient is to crush Roman Catholicism in France first, and then elsewhere." When Masons had previously tried to disavow such excesses, the American Albert Pike wrote: "... it is idle for you to protest. You are Freemasons and you recognize them as Freemasons. You give them countenance, encouragement and support, and you are jointly responsible with them and cannot shirk that responsibility." This is a clear statement of the oneness of Freemasonry throughout the world.
Attacks on the Church
The hostile attitude of Freemasonry can be verified by a brief glance at the leading Masonic publications in the United States and Canada. In some of these, Masonic news itself is of minor importance compared with the detailed and varied attacks made on every conceivable aspect of Catholicism.
These are solid reasons that should make the attitude of the Catholic Church toward Freemasonry reasonable and logical to any thinking person. And it is for such reasons that the Catholic Church has forbidden Catholics to be members of the Masonic Order or any other organization that subscribes to like principles.
Catholics are expected to exercise the same charity and goodwill toward individual Masons that would be manifested toward any other person. Many Masons do not, in practice, carry out the principles that make Freemasonry unacceptable to Catholics. Freemasonry in many places engages in works of charity and philanthropy which are warmly applauded by all Catholics. But even though Catholicism and Freemasonry are irreconcilable, there is no reason why Catholics and Masons should not be personally good neighbors, treating each other with justice and charity.
"The faithful who enroll in Masonic associations are in a state of grave sin and may not receive Holy Communion.... Consequently, neither the excommunication nor the other penalties envisaged have been abrogated." -Cardinal Ratzinger, Sacred Congregation for the Doctrine of the Faith, November 26, 1983
"In the past We have counseled through prophets, your Popes, to forbid all who have joined in My Son's Church, forbid them to become part of freemasonry and the Masons. And why? Because, My children, they are not of God, and if they are not of God, they are the Antichrist. And why are they the Antichrist? Because they worship false idols!
"In the commandments of Your God you will remember: 'I,' said the Lord, 'I am your God; thou shalt not have strange gods before you.' And who are these strange gods of masonry and witchcraft? Isis!
"My children, man has accepted gods of nature! Pagans you have become! You reject your God the Father; you reject the Trinity; and you have dabbled and burned your fingers in the unknown, the darkness of satanism." - Our Lady of the Roses, November 1, 1977
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is not of God
"My children, recognize the faces of evil now loosed in the world. I bring you a sad lesson in knowledge. In the past We have counseled through prophets, your Popes, to forbid all who have joined in My Son's Church, forbid them to become part of freemasonry and the Masons. And why? Because, My children, they are not of God, and if they are not of God, they are the Antichrist. And why are they the Antichrist? Because they worship false idols!" - Our Lady, November 1, 1977
A major
"Speak, My child; speak now, as I have directed you. There is a major law in the Faith of Jesus Christ that non shall enter into secret societies of the Masons. The sons of satan* are rulers, known by the name of Freemasons." - St. Michael, August 21, 1974
* "Indeed each of you knows well the nature, zeal and intention of sects, whether called Masonic or some other name.... [You] cannot doubt but that the present calamity must be attributed to their deceits and machinations for the most part. For from these the synagogue of satan is formed which draws up its forces, advances its standards, and joins battle against the Church of Christ." - Pope Pius IX, Etsi Multa, November 21, 1873
Freemasonry - Synagogue of Satan
"Do not be misguided, O leaders of My Son's House, Church; do not accept the fallacy that masonry is condoned by your God! It is for reason that all of your pastors have condemned freemasonry as a synagogue of satan. Do not fall into the error and the deceit of those who claim brotherhood and goodness, for what god do they recognize in their naturalism? It is not God the Father in Heaven; it is not My Son. It is a deity that will bring destruction to anyone who enters into such a secret society.
"My child, I have given to you, in your possession, a book. Guard it well, so that it will not be taken from you. You understand, My child, the very first page read - and this is a secret society that many of your pastors condone - the very first page reads; 'And Mozart, who was a Roman Catholic, when he weighed the credits of Catholicism and freemasonry, he found that freemasonry was far about Catholicism.' My child and My children, is that the writing that is constructive for your Faith? Is this not an undermining of My Son's Church? 1968 this book was printed, and given into the hands of all members of the lodge." - Our Lady, November 1, 1976
Catholicism: only true religion
"O My children, I will not go into a long discourse now with you about charity and love for your neighbor, but you must understand: you cannot judge your neighbor. You must pray for them. However, you cannot become weak and permissive. You must stand forth as bearers of the light, carriers of the truth. You have been given by your baptism entrance into the only true religion upon earth, the Roman Catholic Church under My Son, Jesus. Though man in his arrogance and pride has forgotten His role and His rule, you must carry it forward. Retain the Faith and the truth in the hearts of mankind." - Our Lady, September 7, 1978
Directives from Heaven... http://www.tldm.org/directives/directives.htm
D13 - Secret Societies PDF
D186 – Excommunication PDF
The pastoral problem of Masonic membership
Declaration on Masonic associations, Sacred Congregation for the Doctrine of the Faith, November 26, 1983
Readers respond to Freemason, Matt C. Abbott, September 4, 2006
Catholicism vs. Freemasonry: Irreconcilable forever
Catholics and Freemasonry religion
Catholic Encyclopedia, "Masonry" (Freemasonry)
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"I must ask you all to read but a few short chapters a day now, the Book of life and love, your Bible. Knowledge must be gained for all the disciples of My Son, for you will be attacked by scientific minds. But do not be concerned what you will say to them when accosted, for the words will be given to you by the Spirit." - Our Lady, April 10, 1976 (Order Form)
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On February 1, 1974, Our Lady said, "My children, know the value of these sacramentals. Guard your children well. You must awaken to the knowledge that you will not be protected without the sacramentals. Guard your children's souls. They must be surrounded with an aura of purity. Remove them if necessary from the sources of contamination, be it your schools or even false pastors."
This Heaven's Personal Protection Packet is available for a donation of $5.00 plus $3.00 shipping and handling. Send $8.00 to TLD Ministries, P.O. Box 40, Lowell, MI 49331. You may use your MasterCard, VISA, or American Express and call 1-616-698-6448. Item # P5 (Order Form)
Your names have been written in Heaven… "It is not by accident that you are called by My Mother, for your names have been written in Heaven.... But with this great grace you have great responsibility to send this Message from Heaven throughout the world, for if you are able to recover just one more for Heaven, an additional star shall be placed in your crown." - Jesus, August 5, 1975
A great obligation to go forward... "It is not by accident that you are called by My Mother, for it is by merit and the prayers that have risen to Heaven for your salvation. For those who have received the grace to hear the Message from Heaven, you have a great obligation to go forward and bring this Message to your brothers and sisters. Do not expect a rest upon your earth, for you will have eternal rest very soon." - Jesus, June 12, 1976
The sin of omission... "The sin of omission shall condemn many to hell, be they layman or Hierarchy. I repeat: not the sin of commission, but the sin of omission will commit many to hell." Our Lady of the Roses, October 6, 1980
We encourage everyone to print or email copies of this web page to all the Bishops and all the clergy. Also, email or send this web page to the news media and as many people as possible.
Email this page to a friend.
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Copyright © These Last Days Ministries, Inc. 1996 - 2011 All rights reserved.
P.O. Box 40 616-698-6448
Lowell, MI 49331-0040
Revised: April 02, 2012


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One Nation under God (8 of 13) by Jerry Vines
This content is part of a series.One Nation under God (8 of 13)
Series: Deuteronomy
Jerry Vines
Deuteronomy 16:18-18:8, 26:1-19
As a way of a reminder to us about what we've been discussing in the study of Deuteronomy, these chapters give us a series of farewell addresses or sermons on the part of Moses as he prepares the people to enter into the Promised Land. He has led them through 40 years of wilderness wandering. They are getting ready to go into the Promised Land; so God, through Moses the servant, is seeking to prepare them and to get them ready for what they are going to face and how to deal with life as it will be lived in the Promised Land.
They are going to have to have some form of government. There are some basic institutions which God has given to human society. They are crucial. They are absolutely essential to human civilization.
One of these is the institution of marriage and the family. That's why marriage and family issues are so very crucial in our culture today. There is an assault being made against the institution of the family. The very definition of the family is being twisted and turned on its head. This is a cause for concern to not only Christian people but to all moral people and to people who understand the essential nature of these basic foundational units in society.
Another one of these foundational units is the institution of government. God established human government. You will find that this is an institution of divine origin. Romans 13, verse 1, says, "Let every soul be subject unto the higher powers. For there is no power but of God; the powers that be are ordained of God." He's saying that the institution of government is ordained of God.
There has to be the institution of government in order that people might live in an orderly, peaceful, efficient society. God has given to us the institution of government.
What Moses is doing is establishing the basic principles of government that will guide the children of Israe ...
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Valentine's Day can be a challenge for men-one misstep can
mean romantic disaster. As for husbands and dads (for whom windows
of amorous opportunity are fewer and further between) - Valentine's
Day is an interpersonal minefield. This week the Dads are here to
lead you through to safety.
WDP12: Valentine's Pitfalls by ChicagoParent
Don't mess up Valentine's Day again this year! Let the Certified
Love Professors* of White Dad Problems help you steer clear of Feb.
14th missteps.
Think the holiday means obligatory sex for you? You're
right! - but don't let your special lady know you think that way.
Do. Not.
The window of opportunity is only open briefly - don't slam it
shut by getting your wife too drunk, too gassy, or by talking to
her. She doesn't need to hear your stupid opinions, she just
needs chocolates and a small amount of booze.
Did you buy a Cupid costume to surprise your sweetie? Yuck.
Return it. (But keep the adult diaper. If you don't need it now
you'll need it soon enough.)
Do not buy a house in February like D.M. did. You'll have a
mortgage and still have to buy dinner. It can wait.
Forget the day was coming? Running to Walgreens to buy a singing
lobster crooning Buster Poindexter's hit? Do not do it.
Buying dinner for her? Maybe don't - you're going to get the
same crummy dinner that restaurant always serves, but at a huge
markup! Make dinner, Mister Lazy.
And finally, if you've been dating that chick for under six
months, no Groupon dates. Especially if it's for something she'll
hate - like paintball or body waxing.
You, sir, are welcome.
Send us a question we can read on the air: [email protected].
Dig the show? There's a lot more to hear by subscribing on
iTunes or visiting whitedadproblems.com.
You can also follow the Dads on Twitter @whitedadprobs and
like their Facebook
*The Cast of White Dad Problems are not Certified Love
Professors. That is not a thing.
White Dad Problems is now The Paternity Test. Head to the new blog page for more great content.
See more of White Dad Problems's stories here.
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