The complete genome of an individual by massively parallel DNA sequencing

Abstract

The association of genetic variation with disease and drug response, and improvements in nucleic acid technologies, have given great optimism for the impact of 'genomic medicine'. However, the formidable size of the diploid human genome, approximately 6 gigabases, has prevented the routine application of sequencing methods to deciphering complete individual human genomes. To realize the full potential of genomics for human health, this limitation must be overcome. Here we report the DNA sequence of a diploid genome of a single individual, James D. Watson, sequenced to 7.4-fold redundancy in two months using massively parallel sequencing in picolitre-size reaction vessels. This sequence was completed in two months at approximately one-hundredth of the cost of traditional capillary electrophoresis methods. Comparison of the sequence to the reference genome led to the identification of 3.3 million single nucleotide polymorphisms, of which 10,654 cause amino-acid substitution within the coding sequence. In addition, we accurately identified small-scale (2-40,000 base pair (bp)) insertion and deletion polymorphism as well as copy number variation resulting in the large-scale gain and loss of chromosomal segments ranging from 26,000 to 1.5 million base pairs. Overall, these results agree well with recent results of sequencing of a single individual by traditional methods. However, in addition to being faster and significantly less expensive, this sequencing technology avoids the arbitrary loss of genomic sequences inherent in random shotgun sequencing by bacterial cloning because it amplifies DNA in a cell-free system. As a result, we further demonstrate the acquisition of novel human sequence, including novel genes not previously identified by traditional genomic sequencing. This is the first genome sequenced by next-generation technologies. Therefore it is a pilot for the future challenges of 'personalized genome sequencing'.

Optimized filtering reduces the error rate in detecting genomic variants by short-read sequencing

“…Another commonly used approach is to apply quality filters that are aimed at selectively removing errors. Every whole-genome sequence reported so far has used filtering to some extent: the most commonly used filters being those that remove sequences with a too-low coverage depth, discard variants with a low-confidence score or eliminate variants located within a cluster of variants 3,7,[10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] . Surprisingly, there is little consensus with respect to which filters should be used and at which threshold they should be applied.…”

The pursuit of genome-wide association studies: where are we now?

“…So far, seven human diploid genomes have been fully sequenced, and some important insights have been gained from these resequencing studies. [145][146][147][148][149][150][151] The most prominent finding from these studies is that, besides SNPs, other genetic variants are also abundant in the human genome. These studies found that in addition to the 3-4 million SNPs, several hundred-thousand of short indels (for example, sizes defined as 3 and 16 bp or less in the Bentley et al 147 and Wang et al 148 study, respectively) are also present in each individual human genome.…”

The discovery of human genetic variations and their use as disease markers: past, present and future

“…As such, the remaining several hundred thousands of indels in the range of several nucleotides to tens of nucleotides, which were identified in the recent whole genome resequencing experiments, do not currently have their own category. [27][28][29][30][31][32][33] For example, Wang et al (2008) 29 found B140 000 indels within 1-3 bp in the Han Chinese YH genome, and B400 000 indels defined from 1 to 16 bp were also detected in the African NA18507 genome by Bentley et al (2008). 30 Perhaps a new category such as 'short indels' needs to be created to fit them in, and those indels between 100 bp to 1 kb should probably be renamed as 'intermediate indels' (Figures 1c and d).…”

“…2 However, the richness of genetic variations in the human genome has recently been further corroborated by the several whole genome resequencing studies, revealing plenty of new SNPs, indels, CNVs and other structural variations. [27][28][29][30][31][32][33] The technological developments have facilitated and accelerated the process of identifying genetic variations, especially with the arrival of next generation sequencing technologies, which have made whole genome resequencing and the 1000 Genomes Project feasible. [53][54][55] In recent years, many studies have been done to directly examine the associations of CNVs with complex diseases using SNP genotyping arrays.…”

“…77 As a substantial fraction of entries in the dbSNP has not been validated in population-based studies, one has to bear in mind that not all the entries in the dbSNP are necessarily SNPs, as the name of database implies. As such, the several hundred thousand 'new SNPs' identified by whole genome resequencing studies [27][28][29][30][31][32][33] should probably be considered as 'new SNVs' instead, until their population frequency information is available (Figure 3a). The distinction between SNPs and SNVs should be emphasized to avoid misleading.…”