2.2. Target selection and generation of customized target capture
For sequencing, we selected 988 genes implicated in the pathways of aging and longevity in humans. The rationale for selection and their criteria are described in Section 3.1 under Results. The complete list of thecorrectly detect the variants. Further analysis showed that the confirmed nsSNPs were present in a heterozygous state in the centenarians, with allele frequencies ranging from 0.10 to 0.47. To test whether these nsSNPs were enriched in centenarians compared to controls, we genotyped them in a larger sample set consisting of 192 centenarians and 192 controls. Association analysis showed that three nsSNPs were significantly associated with longevity after Bonferroni correction (p < 5.13 × 10-4), including rs145072688 (p.Arg87His) in GPR50, rs1801133 (p.Cys677Tyr) in MTHFR, and rs17185536 (p.Arg1174Gln) in FLT1. These findings suggest that these novel nsSNPs may play a role in human longevity and warrant further investigation.

2.3. Library preparation, target enrichment, and sequencing
We isolated DNA from immortalized B-lymphocytes established from six centenarians and generated libraries for Illumina sequencing. Each library was indexed with a unique barcode, which allowed for target capture and enrichment of pooled libraries and multiplex sequencing. The library preparation and enrichment methods were performed according to the Agilent Illumina Paired-End Sequencing Platform Library Prep Protocol, with the following modifications: (1) genomic DNA was sheared with Covaris system for 250 bp; (2) Illumina adapters were replaced with custom-made, indexed adapters supplied by IDT DNA (see Supplementary Table S2 for adapter sequence information); (3) size-selection and DNA elution were performed by precast E-gel (Invitrogen) SizeSelect 2% with E-gel precast gel cassette, and fragments with the desired size (300-350 bp) were eluted with 25 ml water; (4) for the pre-capture enrichment PCR, DNA was amplified in eight cycles; (5) post-enriched DNA libraries were combined in equal amounts to a total quantity of 1 mg; (6) the hybridization mixture was incubated for 24 h at 65°C to minimize evaporation. Target-enriched libraries were sent to Axeq Technologies and sequenced paired-end on the Illumina HiSeq2000 with cluster kit v2 according to the manufacturer's protocol. Reads generated were 101 bp in length.

Accumulation of DNA damage and mutations has been considered an important causal factor in age-related deterioration and death (Garinis et al., 2008; Hoeijmakers, 2009). This concept is in keeping with the proposed evolutionary basis of aging, i.e., the declining force of natural selection with age, which does not predict maximization of cellular maintenance and repair (Gavrilov and Gavrilova, 2002). DNA damage continuously occurs by both endogenous and exogenous sources, such as reactive oxygen species (ROS) produced as byproducts of metabolism, UV light, and ionizing radiation. DNA damage is known to cause aging-related cellular degeneration and diseases. The insulin/insulin-like growth factor I (IGF-I) signaling pathway modulates DNA damage and stress response and is well-established as a conserved pathway of aging (Kenyon, 2010; Tazearslan et al., 2012). Mutations in insulin/ Please cite this article in press as: Han, J., et al., Discovery of novel non-synonymous SNP variants in 988 candidate genes from 6 centenarians by target capture and next-generation sequencing. Mech. Ageing Dev. (2013), http://dx.doi.org/10.1016/ J. Han et al./Mechanisms of Ageing and Development xxx (2013) xxxxxx 

2.4. Data analysis
We used the Agilent eArray to design and assess coverage across the target genomic regions of bait libraries. Design efficiency from eArray was 99%, calculated as the covered fraction of target bases in the oligo design (6,229,389 bp), out of the total submitted target regions (6,291,136 bp). The final customized target capture contained 5,692,804 bp covering the target regions of the 988 candidate genes.

Sequencing reads were aligned to the human genome revision hg19 (Lander et al., 2001) using BWA version 0.5.9 (Li and Durbin, 2009), with setting "-q 17" provided when running the "aln" command. Following alignment, we utilized Picard [http://picard.sourceforge.net] to detect potential PCR duplicates and to calculate on-target statistics. Subsequent processing was performed using the Genome Analysis Toolkit (GATK) (McKenna et al., 2010). To call variants, we first preprocessed the dataDetect heterozygote calls in the same individuals in which the novel nsSNPs were initially discovered, as well as in a larger number of controls (n = 410) and centenarians (n = 390) (Fig. 1B). 

3.3. Genotyping and association analysis of novel nsSNPs in 410 controls and 390 centenarians 

2.5. Genotyping 

To validate novel nsSNPs discovered in six centenarians, we designed iPLEX MassArray assays (Sequenom) using the web-based assay design suite program on the mysequenom website (http://www.mysequenom.com). From a total of 89 novel nsSNPs, 67 nsSNPs were designed for iPLEX assays in four separate multiplexing groups based on the high quality of peaks visualized in the MassARRAY Typer software after performing MassArray. Using the iPLEX assays, genotyping was performed in 410 controls and 390 centenarians at the Genomics Shared Facility at Albert Einstein College of Medicine.