Scientists have added to the complex and controversial tales of origin surrounding the Turin Shroud, by revealing the myriad locations of the DNA and pollen that are present on the cloth. 
 
 The length of linen is highly contentious. Some Christians believe the fabric – which is kept in the Cathedral of Saint John the Baptist in Turin - is the burial shroud of Jesus of Nazereth, dating back over 2,000 years. 
 
 However,previous scientific studies have suggested that the cloth, which appears to be imprinted with the image of a man, may in fact be from the 13th or 14th century - centuries after Jesus is believed to have died. 
 
 Now, a new study by Italian researchers published in the prestigious science journal Nature gives a further insight into the potential origins of the cloth. 
 
 To make their findings for the paper entitled 'Uncovering the sources of DNA found on the Turin Shroud', the team behind the study sequenced the DNA of pollen and other dust from the cloth. 
 
 By doing so, they were able to pinpoint the biogeographic origin of the people who came into contact with the shroud. 
 
 The authors found traces on the parchment from people and pollen from around the world, including Europe, south Asia, eastern Africa and the Middle East. 
 
 Researchers said that the findings could indicate that that the shroud made its "historic [...] presumed journey" from the Near East and was taken around the world before being kept in Italy; or that it has its origins in medieval Europe where and was contaminated by those who travelled to it. 
 
 In a further twist, the study indicated that it may have been manufactured in India. ||||| Collection of TS Samples for DNA analyses 
 
 The samples used in this study for DNA investigations are sub-sets of the dust particles vacuumed from the back of the Turin Shroud (TS), which were kindly provided in 2010 by Giulio Fanti (Dept. of Industrial Engineering, University of Padua, Italy), who in turn received them personally from Giovanni Riggi di Numana in 200612,18 (see also Supplementary Document S1). In particular, four samples were originally collected on filters in 1978 from four distinct areas of the back of TS, corresponding to the hands (filter E), face (filter F), feet (filter G) and glutei (filter H) of the TS male image12. An additional sample was collected in 1988, deriving specifically from the corner area (filter I) of TS, which was used for radiocarbon dating8,12 (for details, see Supplementary Figure S2, panels A–C). 
 
 The dust samples collected on these filters were immobilized on adhesive tapes as previously described6,13 and consisted of a variety of particles. In fact, optical microscope observations of filters E-I had previously revealed the presence of pollen grains, cell debris and other minuscule organic specimens, such as plant-derived fibers and blood-like clots6,7,13. 
 
 The five pieces of adhesive tape containing TS dust particles collected on filters E, F, G, H and I were supplied as portions of the original samples (ranging in size from 5 × 10 mm to 10 × 30 mm, see the schematic representation in Supplementary Figure S2, panel D), each attached to a sterile microscope slide. Each piece of adhesive tape was manually cut using a scalpel and tweezers under a stereomicroscope into tiny sub-portions of dimensions on the order of a few square millimeters (~5 × 5 mm each); the pieces were then individually transferred into 1.5-ml sterile microtubes. A total of 2 to 12 square portioned specimens of the pieces of adhesive tape were used for independent genomic DNA extractions; the DNA samples from the individual specimens were kept separate for PCR amplifications. 
 
 PCR amplicons obtained from each of the cpDNA regions were sub-cloned and used individually for DNA sequencing, whereas PCR amplicons obtained from each of the mtDNA regions were pooled for 454 sequencing reactions. In particular, the DNA amplicons from each of the 2, 4 or 6 specimens associated with internal filters E, F, G and H were pooled together into single samples, whereas the DNA amplicons from external filter I were divided into two samples of 6 specimens each (named I and I R and taken as independent biological replications; Supplementary Figure S2). This pooling strategy was imposed by the different sizes of the pieces of adhesive tape to be analyzed for each of the five TS filters and by the fact that dust particles were demonstrated to be much more abundant on the tape representing the external filter than on those for the internal filters6,13. 
 
 All manipulation steps were performed aseptically under laminar flow hood conditions using autoclaved disposables and buffers that were filter-sterilized. In particular, we used sterile materials and all standard procedures with internal negative controls to avoid, or eventually discover, contaminations from the operators and the environment. The entire mitochondrial genomes of the three operators who came in contact with the Shroud samples were completely sequenced and all mtDNA sequences that we obtained from the TS samples and could be theoretically attributed to operator contamination (Supplementary Table S3) were excluded from our final results. As for possible environmental contaminations, none of the large varieties of plant sources detected in the Shroud (Supplementary Table S1) through the analysis of nuclear ITS, chloroplast genes or cpDNA sequences are grown or studied in our facilities. 
 
 Genomic DNA extraction 
 
 Specimens from the pieces of adhesive tape with the TS dust particles were used individually for genomic DNA extraction to avoid any loss of TS material and to perform replicate experiments for each TS filter. 
 
 All genomic DNA extractions were performed with the QIAamp®DNA Investigator commercial kit (Qiagen) by applying the protocol originally developed for laser-microdissected specimens, with some modifications. All manipulation steps were performed under laminar flow hood conditions using DNA- and DNase-free, disposable autoclaved materials and filter-sterilized buffers. Immediately after collection, sub-samples were transferred to a 1.5-ml tube containing a pre-warmed mixture composed of 40 μl of ATL buffer and 20 μl proteinase K. Each sample was then mixed by pulse-vortexing and incubated at 56 °C for 16 hours under constant agitation. After this step, 100 μl of the AL buffer, containing 2 μg of carrier RNA, was added, and the solution was mixed by pulse-vortexing for 15 sec. Then, 100 μl absolute ethanol was added, and the solution was mixed thoroughly by pulse-vortexing for 15 sec and then incubated for 5 min at room temperature. The next steps, which consisted of DNA immobilization to a silica membrane and wash steps to clean the isolated DNA from salts and impurities, were conducted by following the manufacturer’s suggestions. Elution of the DNA immobilized by the silica membrane was performed in two steps by using 15 μl ATE buffer at each elution step and by extending the incubation step to 10 min at room temperature. 
 
 To identify human genetic lineages and plant taxonomic entities, specific plant DNA and human mitochondrial DNA regions were designated for PCR amplification and sequencing. 
 
 PCR amplification and sequencing of plant and human DNA sequences 
 
 PCR amplifications of plant DNA sequences were performed with primer combinations designed using nuclear (rDNA intergenic transcribed spacers, ITS) and chloroplast targets (cpDNA barcodes RuBisCO or rbcL, trnH-psbA, and trnL-intron) (Supplementary Table S4), following already available protocols19,20. Briefly, the reactions were performed in a total volume of 20 μl that included 2 μl of 10X reaction buffer, 1 mM MgSO 4 , 0.3 mM dNTPs, 0.25 U of Platinum® Pfx DNA Polymerase (Life Technologies), 0.3 μM of primer mix and 2.5 μl of eluted DNA solution. The reactions were performed in a 9700 Thermal Cycler (Applied Biosystems) using a temperature profile that consisted of an initial denaturation step of 10 min at 95 °C followed by 50 cycles of 1 min at 95 °C, 30 sec at 55 °C, 1 min at 68 °C and a final step of 10 min at 68 °C. 
 
 The same conditions were adopted for PCR amplifications of human sequences corresponding to three distinct mtDNA loci (MT-DLOOP with the hypervariable segments MT-HV1 and MT-HV2, MT-CO1, and MT-ND5) using specific primer pairs (Supplementary Table S4). 
 
 The amplicons were sub-cloned by ligation into the TOPO-blunt cloning vector (Life Technologies) and transformed into chemically competent one-shot TOP10 bacterial cells (Life Technologies). Clones were plated on LB plates (1.5% agar, 50 μg/mL ampicillin, 40 μg/ml X-Gal), and transformed colonies were selected by Colony-PCR. Amplification reactions were performed in a total volume of 20 μl including 2 μl of 10X reaction buffer, 1.5 mM MgCl 2 , 300 μM dNTPs, 1.5 U of BIOTaq DNA polymerase (BIOLINE), 0.2 μM of M13For (5′-GTAAAACGACGGCCAG-3′) and M13Rev (5′-CAGGAAACAGCTATGAC-3′) primers. Positive colonies were sequenced using an ABI3100 automated sequencer (Applied Biosystems). 
 
 Both nuclear ITS sequences and DNA barcodes were used to identify plant and fungal species21,22, querying the Barcode of Life Data BOLD Systems v. 3 (http://www.boldsystems.org/index.php/IDS_OpenIdEngine). Sequence similarity searches in nucleotide collections (nt) and with non-redundant protein sequences (nr) were also performed with blastn/x programs of the Basic Local Alignment Search Tool BLAST v. 2.2.30+ (http://blast.st-va.ncbi.nlm.nih.gov/Blast.cgi) using default parameters. All plant cpDNA and ITS sequences deriving from PCR amplicons were verified through replicated sequencing of both strands and deposited in GenBank with accession numbers JQ007354-JQ007431 and JQ082521-JQ082524. The human mtDNA sequences, as clusters deriving from the assembly of reads generated by 454 sequencing, were deposited in GenBank with accession numbers KP126143-KP126230. 
 
 For each plant sequence, we collected information regarding: i) the TS location source; ii) the GenBank accession number of the most similar sequence; iii) the Linnaean name of the species, including the botanical family and its common name; iv) the name and length of the target DNA sequence; v) the center of origin and geographical distribution of the species, along with information on plant type and/or common use (for details, see Supplementary Table S1). From an experimental point of view, it should be noted that some species were identified in biological replicates by using different specimens of the same filter as well as different filters but were also assessed by technical replicates according to amplicons from different genetic targets. In addition, other species were identified by performing independent experiments, meaning that the PCR analyses were conducted at different times using DNA samples isolated at different times from the same filters/dust samples. All these cases are pivotal experimental validations and demonstrate the robust assignments of plant species (or genus). 
 
 PCR amplification of human mtDNA and preparation of libraries for pyrosequencing 
 
 Amplifications of target mtDNA sequences prior to pyrosequencing were performed as described in the previous paragraph using specific primer sets (Supplementary Table S4). It is worth mentioning that the successful amplification of human mtDNA fragments ranging in size from 419 to 576 bp (as well as plant nuclear and chloroplast DNAs from 229 to 622 bp) such as the ones that we amplified, would be mostly possible from undamaged or slightly damaged DNA templates, which is unexpected when dealing with very ancient DNA specimens. However, the size of ancient DNA fragments, in addition to age, is influenced by many additional factors such as environmental conditions (e.g., temperature, moisture and pH) and mode of preservation (e.g., museum specimens vs. freshly excavated remains). In brief, DNA degradation becomes more effective only over long intervals, but is de facto unpredictable over shorter time spans such as those postulated for the DNA traces on the TS and when considering the numerous copies of extra-nuclear DNA molecules23,24,25,26. 
 
 PCR products originating from the amplification of multiple samples were pooled (Supplementary Table S4) and purified with QIAquick PCR Purification Kit (QIAGEN) by following the recommendations of the supplier. Next, the amplicons of the three different mtDNA target regions were pooled together, as reported in Supplementary Table S5, and purified with the Agencourt AMPure XP procedure (Beckman Coulter) by following the recommendations of the supplier. Quantification of the pooled amplicons was performed with a Nanodrop fluorometer ND 3300 (Thermo Scientific). 
 
 Libraries were prepared starting from 100 ng of purified PCR products using Rapid Library Preparation Method (Series GS FLX+; Roche 454 sequencing), starting from step 3.2: Fragment End Repair (thus avoiding the step 3.1, DNA Fragmentation by Nebulization). Single libraries were tagged with univocal MIDs (Supplementary Table S6). Emulsion PCR and 454 sequencing were carried out according to the manufacturer’s instructions on the Roche 454 FLX Titanium platform. Library sequencing was carried out using a Roche 454 GS Junior System. 
 
 Analysis of sequence reads, variant calling and cluster assembly 
 
 Sequence reads were de-multiplexed based on their MID sequence with the Roche 454 software SFFfile. The number of sequenced and aligned reads referring to the single libraries are reported in Supplementary Table S7. 
 
 Mapping of the sequence reads to the H. sapiens mitochondrial genome (gb|NC_012920.1) was performed with the software CLC Genomics Workbench version 5.05 with defaults parameters. Mapping of the sequence reads originating from each library was used to estimate the average coverage for each target region. Background noise in the sequences was filtered with a program developed ad hoc that analyzes the composition in words (k-mer) of the produced sequences and masks the words having a frequency lower than the value set as the cutoff. For this step, the length of the k-mers was set to 7, and the minimum frequency parameter was set to 5. After the masking of rare variants, the sequences were clustered with the software CD HIT v.4.5.427 by setting a threshold of percent identity equal to 99%. The average coverage of the mtDNA target regions for each single pool and target region is summarized in Supplementary Table S7. 
 
 Sequence clusters with a length greater than 200 bp were aligned to the H. sapiens mitochondrial genome (gb|NC_012920.1) using the program “bwa”28 with default parameters. In this step, each pool of sequences was independently aligned against the reference sequence17. The alignment files were saved as standard SAM files. Variant calling was performed on alignments of sequences representative of each cluster with the software PASS29. For each cluster, the haplotype was defined as the collection of substitutions (either transversions or transitions) that emerged in the variant calling process. Insertions and deletions were not taken into account. 
 
 A pre-filtering step for estimation of the mtDNA haplogroups represented in our cluster sequences was performed with the software HaploGrep30, which allows the automatic assignment of haplogroups according to Phylotree31. Preliminary assignments of haplogroups to sequence clusters displaying the highest sequence coverage were manually verified. The frequency of each haplogroup was estimated by the number of sequences contained in each cluster assigned to that specific haplogroup. The distribution of haplogroups in the different samples was estimated as above, but by counting the sequences according to the library information. The assembled human mtDNA sequences were deposited in GenBank with accession numbers KM655881-KM655934 (for details, see Supplementary Table S8). ||||| Editor's Note: This story was updated at 1:55 p.m. E.T. 
 
 Is it a medieval fake or a relic of Jesus Christ? A new analysis of DNA from the Shroud of Turin reveals that people from all over the world have touched the venerated garment. 
 
 "Individuals from different ethnic groups and geographical locations came into contact with the Shroud [of Turin] either in Europe (France and Turin) or directly in their own lands of origin (Europe, northeast Africa, Caucasus, Anatolia, Middle East and India)," study lead author Gianni Barcaccia, a geneticist at the University of Padua in Italy and lead author of the new study describing the DNA analysis, said in an email. "We cannot say anything more on its origin." 
 
 The new findings don't rule out either the notion that the long strip of linen is a medieval forgery or that it's the true burial shroud of Jesus Christ, the researchers said. 
 
 Long-standing debate 
 
 On its face, the Shroud of Turin is an unassuming piece of twill cloth that bears traces of blood and a darkened imprint of a man's body. Though the Catholic Church has never taken an official stance on the object's authenticity, tens of thousands flock to Turin, Italy, every year to get a glimpse of the object, believing that it wrapped the bruised and bleeding body of Jesus Christ after his crucifixion. [Religious Mysteries: 8 Alleged Relics of Jesus] 
 
 According to legend, the shroud was secretly carried from Judea in A.D. 30 or 33, and was housed in Edessa, Turkey, and Constantinople (the name for Istanbul before the Ottomans took over) for centuries. After crusaders sacked Constantinople in A.D. 1204, the cloth was smuggled to safety in Athens, Greece, where it stayed until A.D. 1225. 
 
 However, the Catholic Church only officially recorded its existence in A.D. 1353, when it showed up in a tiny church in Lirey, France. Centuries later, in the 1980s, radiocarbon dating, which measures the rate at which different isotopes of the carbon atoms decay, suggested the shroud was made between A.D. 1260 and A.D. 1390, lending credence to the notion that it was an elaborate fake created in the Middle Ages. (Isotopes are forms of an element with a different number of neutrons.) 
 
 But critics argued that the researchers used patched-up portions of the cloth to date the samples, which could have been much younger than the rest of the garment. 
 
 What's more, the Gospel of Matthew notes that "the earth shook, the rocks split and the tombs broke open" after Jesus was crucified. So geologists have argued that an earthquake at Jesus' death could have released a burst of neutrons. The neutron burst not only would have thrown off the radiocarbon dating but also would have led to the darkened imprint on the shroud. 
 
 Global traveler? 
 
 In the current study, Barcaccia and his colleagues analyzed dust that they vacuumed from the shroud that contained traces of both plant and human DNA. 
 
 The plant DNA came from all over the world, the researchers reported Oct. 5 in the journal Scientific Reports. European spruce trees; Mediterranean clovers, ryegrasses and plantains; North American black locust trees; and rare East Asian pear and plum trees all left their mark on the cloth. 
 
 The team also sequenced the human mitochondrial DNA (DNA passed from mother to child) found in dust from the shroud. The genetic lineage, or haplotype, of the DNA snippets suggested that people ranging from North African Berbers to East Africans to inhabitants of China touched the garment. 
 
 Still, the strongest genetic signals seemed to come from areas in and around the Middle East and the Caucasus — not far from where Jesus was buried, and consistent with the early folklore surrounding the object. [The 10 Most Controversial Miracles] 
 
 "One of the most abundant human mitochondrial haplotypes, among those discovered on the shroud, is still very rare in western Europe, and it is typical of the Druze community, an ethnic group that has some origin in Egypt and that lives mainly in restricted areas between Syria, Jordan, Lebanon, Israel and Palestine," Barcaccia told Live Science in an email. 
 
 The oldest DNA snippets (which tend to be shorter because DNA breaks down over time) are found in many places on the shroud, and come from genetic lineages typically found only in India, Barcaccia said. That finding suggests that the shroud was manufactured in India before somehow making its way to Europe, as Indians had little contact with Europeans at the time of its origin. 
 
 "In my opinion, it is hard to believe that in the past centuries, in a historical interval spanning the medieval period, different subjects — such as priests, monks or nuns, as well [as] devotees and other subjects of Indian ancestry — have had the possibility to come in contact with the shroud in France and/or Turin," Barcaccia said. 
 
 Unsettled question 
 
 But the new results don't settle questions about the shroud's authenticity, said Hugh Farey, editor of the British Society of the Turin Shroud newsletter. [Who Was Jesus, the Man?] 
 
 As far as the plant DNA goes, "they've done a good job, and they've identified a number of species that mean, broadly speaking, nothing at all," Farey told Live Science. 
 
 The new study suffers from the same issues that made past studies of pollen on the shroud unreliable, said Renée Enevold, a geoscientist at the Moesgaard Museum in Denmark who has analyzed ancient pollen in the past. 
 
 "The plant DNA could be from many sources, and there is no way of finding the right source," Enevold told Live Science in an email. "Also, the sub-genus level of taxon that has been reached is not near enough to the species level that is needed to determine the area of origin for each plant." 
 
 The researchers also mistakenly relied on an interpretative method that is used to analyze thousands of grains of pollen in a lake, she said. In that environment, the conditions that led to the deposition of pollen — rain and wind, for instance — are known. In contrast, there are so many unknowns when it comes to describing how dust settled onto the shroud. 
 
 "It is very bold and completely wrong to use the same interpretational approach on the presence of DNA — or just a few pollen grains, for that matter — on a shroud that has been man-handled for decades," Enevold said. 
 
 Given that the cloth was publicly displayed for centuries, it's not surprising that so many people touched it, Farey added. "Apart from ruling out the United States of America as the source for the shroud, it leaves just about everything else open," Farey said. 
 
 As for the possible Indian manufacture, it's just as likely that Indian DNA got onto the object during its 20th-century testing, he said. To truly determine where the cloth was manufactured, the researchers would need to analyze the DNA from the flax seeds used to make the linen shroud, which was not done, he added. 
 
 Still, Farey said he's about 40 percent convinced the shroud is authentic and about 60 percent inclined to believe it is a forgery. 
 
 "There is a pretty substantial amount of evidence on both sides," Farey said. "So the proper thing to do is to maintain an open mind at the moment." 
 
 However, using DNA analysis and more sophisticated scientific techniques could ultimately settle the question, Farey said. For instance, geologists can now determine the origin of rock with incredible precision, by analyzing its ratio of isotopes of certain elements. If researchers can one day figure out how to test the isotopes in the limestone dust found on the shroud, they could say with greater certainty whether the shroud was ever in Jerusalem, he said. 
 
 Editor's Note: This story was edited to note that the crusaders, not the Ottomans, sacked Constantinople in A.D. 1204. The Ottomans conquered the city in A.D. 1453. 
 
 Follow Tia Ghose on Twitterand Google+. Follow Live Science @livescience, Facebook & Google+. Original article on Live Science.
– The mysteries of the Shroud of Turin are proving to be very stubborn in the face of modern technology. An Italian team analyzed DNA from the relic some believe is the burial cloth of Jesus but failed to find conclusive proof of its origins, the Independent reports. The testing revealed traces of people and pollen from many parts of the world, meaning it that could be a medieval forgery—or that its journey to Europe could indeed have begun in Jerusalem around 30 or 33AD. The results show that over the centuries, the shroud came in contact with many "different types of natural and anthropological environments," according to the researchers, whose study is published in the journal Nature. One find that will surprise skeptics: One of the most common snippets of mitochondrial DNA was from the Middle East. The type is "rare in western Europe, and it is typical of the Druze community, an ethnic group that has some origin in Egypt and that lives mainly in restricted areas between Syria, Jordan, Lebanon, Israel, and Palestine," lead researcher Gianni Barcaccia tells LiveScience, which notes that other experts consider some of the methods used unreliable. Another find: Barcaccia says some of the oldest DNA on the cloth was from India, meaning the shroud may have been made there. (An earlier study suggested the shroud is real—and was created by a huge earthquake.)