Sequence and structural variation in a human genome uncovered by short-read, massively parallel ligation sequencing using two-base encoding

Abstract

We describe the genome sequencing of an anonymous individual of African origin using a novel ligation-based sequencing assay that enables a unique form of error correction that improves the raw accuracy of the aligned reads to >99.9%, allowing us to accurately call SNPs with as few as two reads per allele. We collected several billion mate-paired reads yielding ;183 haploid coverage of aligned sequence and close to 3003 clone coverage. Over 98% of the reference genome is covered with at least one uniquely placed read, and 99.65% is spanned by at least one uniquely placed matepaired clone. We identify over 3.8 million SNPs, 19% of which are novel. Mate-paired data are used to physically resolve haplotype phases of nearly two-thirds of the genotypes obtained and produce phased segments of up to 215 kb. We detect 226,529 intra-read indels, 5590 indels between mate-paired reads, 91 inversions, and four gene fusions. We use a novel approach for detecting indels between mate-paired reads that are smaller than the standard deviation of the insert size of the library and discover deletions in common with those detected with our intra-read approach. Dozens of mutations previously described in OMIM and hundreds of nonsynonymous single-nucleotide and structural variants in genes previously implicated in disease are identified in this individual. There is more genetic variation in the human genome still to be uncovered, and we provide guidance for future surveys in populations and cancer biopsies.[Supplemental material is available online at

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

“…Using the same approach, hundreds of inversions were also uncovered by whole genome resequencing studies; for example, 91 and 415 inversions were detected in the African NA18507 genome and Korean SJK genome, respectively. 32,106 Although the progress in the discovery of inversions is moving at a slower pace than CNVs, there is already evidence to support their roles in human diseases. 107,108 Loss of heterozygosity and homozygosity Copy neutral LOH defines a continuous stretch of DNA sequence without heterozygosity.…”

Human genome at ten: The sequence explosion

“…G r e e n p l a n t s Metagenomes (multiple species) P r o t o z o a N o n -h u m a n v e r t e b r a t e s H u m a n s I n v e r t e b r a t e s First drafts of two composite haploid human genomes 1,2 Human Genome Project completed 3 Two Korean males including Seong-Jin Kim 9,10 , Stephen Quake 11 , another cancer genome 12 , George Church, a Yoruban female, another male 13 , and four others [14][15][16] A glioma cell line 17 , Inuk 18 , !Gubi and Archbishop Desmond Tutu 19 , James Lupski 20 , and a family of four 21 James Watson 5 , a woman with acute myeloid leukemia 6 , a Yoruba male from Nigeria 7 and the first Asian genome 8…”