Whole-genome haplotyping using long reads and statistical methods

Abstract

Rapid growth of sequencing technologies has greatly contributed to increasing our understanding of human genetics. Yet, in spite of this growth, mainstream technologies have been largely unsuccessful in resolving the diploid nature of the human genome. Here we describe statistically aided long read haplotyping (SLRH), a rapid, accurate method based on a simple experimental protocol that requires potentially as little as 30 Gbp of sequencing in addition to a standard (50x coverage) whole-genome analysis for human samples. Using this technology, we phase 99% of single-nucleotide variants in three human genomes into long haplotype blocks of 200 kbp to 1 Mbp in length. As a demonstration of the potential applications of our method, we determine allele-specific methylation patterns in a human genome and identify hundreds of differentially methylated regions that were previously unknown. Such information may offer insight into the mechanisms behind differential gene expression.

Haplotype-phased synthetic long reads from short-read sequencing

“…In contrast to dilution-based synthetic read approaches [17][18][19], the intramolecular circularization step in our protocol makes it possible to prepare a library in a single tube. We further exploited this property to allow multiple samples to be combined and prepared in the same mixture, yielding considerable savings in cost and time.…”

“…"True" long read technologies such as Pacific Biosciences SMRT sequencing and nanopore technologies [10,11] have proven invaluable in select applications, but require extensive computational error correction [12,13]. As an alternative, sample preparation protocols have emerged that enable long "synthetic" reads to be constructed from conventional short reads [14][15][16][17][18][19][20][21]. However, these approaches have been limited by generalizability, cost, throughput, or synthetic read length.…”

Read Clouds Uncover Variation in Complex Regions of the Human Genome

“…An alternative approach used in LFR (Peters et al 2012), CPTseq (Amini et al 2014), and Illumina TruSeq Synthetic Long-Reads (previously known as Moleculo) (Kuleshov et al 2014) utilizes accurate short-read sequencing of long DNA fragments in order to obtain long-range information at high nucleotide accuracy. The Illumina TruSeq protocol is able to produce 10-kbp long reads, retaining the benefits of the highly accurate and cost-effective Illumina technology (Kuleshov et al 2014) and enabling human genome phasing (Kuleshov et al 2014) and de novo assembly of complex genomes (Voskoboynik et al 2013;McCoy et al 2014).…”

“…The Illumina TruSeq protocol is able to produce 10-kbp long reads, retaining the benefits of the highly accurate and cost-effective Illumina technology (Kuleshov et al 2014) and enabling human genome phasing (Kuleshov et al 2014) and de novo assembly of complex genomes (Voskoboynik et al 2013;McCoy et al 2014).…”

“…Under Illumina's synthetic long-read protocol, DNA sequencing libraries are prepared as follows: First, the genomic DNA is sheared into long (≥10 kbp) fragments and ligated with amplification adapters at both ends; second, these molecules are diluted into wells so that each well receives only a small fraction (1%-2%) of the genome; third, molecules are amplified, sheared into short fragments, and uniquely barcoded within each well (Kuleshov et al 2014). The individual wells are then pooled and sequenced together.…”