Single-molecule sequencing of an individual human genome

Abstract

Recent advances in high-throughput DNA sequencing technologies have enabled order-of-magnitude improvements in both cost and throughput. Here we report the use of single-molecule methods to sequence an individual human genome. We aligned billions of 24- to 70-bp reads (32 bp average) to ~90% of the National Center for Biotechnology Information (NCBI) reference genome, with 28× average coverage. Our results were obtained on one sequencing instrument by a single operator with four data collection runs. Single-molecule sequencing enabled analysis of human genomic information without the need for cloning, amplification or ligation. We determined ~2.8 million single nucleotide polymorphisms (SNPs) with a false-positive rate of less than 1% as validated by Sanger sequencing and 99.8% concordance with SNP genotyping arrays. We identified 752 regions of copy number variation by analyzing coverage depth alone and validated 27 of these using digital PCR. This milestone should allow widespread application of genome sequencing to many aspects of genetics and human health, including personal genomics.

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.…”

“…Therefore, the DNA sequence of any two genomes is estimated to be about 99.9% identical, and the 0.1% genetic variations that are mainly comprised of SNPs, are believed to be responsible for the phenotypic differences, such as physical traits (for example, height, hair and eye colors), disease susceptibility and drug responses, among individuals in populations. However, the finding of thousands of CNVs that collectively encompass hundreds of megabases of the genome [8][9][10] and the numerous short indels that are identified by whole genome resequencing studies [27][28][29][30][31][32][33] have thrown doubts to the estimation of '99.9% similarity' . The DNA sequences of individuals within and between populations are genetically more diverse and varied than previously thought.…”