Whole-genome haplotyping by dilution, amplification, and sequencing

Abstract

Standard whole-genome genotyping technologies are unable to determine haplotypes. Here we describe a method for rapid and cost-effective long-range haplotyping. Genomic DNA is diluted and distributed into multiple aliquots such that each aliquot receives a fraction of a haploid copy. The DNA template in each aliquot is amplified by multiple displacement amplification, converted into barcoded sequencing libraries using Nextera technology, and sequenced in multiplexed pools. To assess the performance of our method, we combined two male genomic DNA samples at equal ratios, resulting in a sample with diploid X chromosomes with known haplotypes. Pools of the multiplexed sequencing libraries were subjected to targeted pull-down of a 1-Mb contiguous region of the X-chromosome Duchenne muscular dystrophy gene. We were able to phase the Duchenne muscular dystrophy region into two contiguous haplotype blocks with a mean length of 494 kb. The haplotypes showed 99% agreement with the consensus base calls made by sequencing the individual DNAs. We subsequently used the strategy to haplotype two human genomes. Standard genomic sequencing to identify all heterozygous SNPs in the sample was combined with dilution-amplification-based sequencing data to resolve the phase of identified heterozygous SNPs. Using this procedure, we were able to phase >95% of the heterozygous SNPs from the diploid sequence data. The N50 for a Yoruba male DNA was 702 kb whereas the N50 for a European female DNA was 358 kb. Therefore, the strategy described here is suitable for haplotyping of a set of targeted regions as well as of the entire genome.genotype | next generation sequencing | phasing

In vitro, long-range sequence information for de novo genome assembly via transposase contiguity

“…We also attempted to apply fragScaff to data generated with fosmid (Kitzman et al 2011) or long fragment read (LFR) (Kaper et al 2013) dilution pools, rather than CPT-seq. However, this resulted in low link counts of poor accuracy (N50 improvement: 1.33 and 1.53; join accuracy: 71.6% and 34.0%; sequence joined: 38.9% and 46.6% for fosmid and LFR, respectively), predominantly due to the reduced pool counts, which restrict the number of coincidences between pool content on which this method is based (fosmid: 288, LFR: 96 versus CPT-seq: 9216).…”

“…Fosmid libraries were previously generated for Adey et al (2013). Long fragment read (LFR) libraries were generated on CEU gDNA using methods outlined in Kaper et al (2013).…”

“…Sequence reads corresponding to each of the 9216 ''virtual compartments'' (also referred to as indexed pools) provide sparse, shotgun representation of the high molecular weight genomic DNA fragments that were transposed within that indexed pool (Fig. 1C), analogous to fosmid (Kitzman et al 2011) or in vitro dilution pools (Kaper et al 2013) but with a ;1003 higher effective number of pools. With appropriate dilution of the input genomic DNA, these indexed pools are ''subhaploid,'' in that the lengths of fragments contained in each pool sums to ;5% to 10% of full genome length.…”

Advancements in Next-Generation Sequencing

“…The formal definition of N50 is the length for which the collection of all contigs of that length or longer contains at least half of the sum of the lengths of all contigs, and for which the collection of all contigs of that length or shorter also contains at least half of the sum of the lengths of all contigs. Illumina independently published an approach that targeted a 1-Mb region of the X chromosome, phasing more than 95% of SNPs and deriving haplotype blocks of hundreds of kilobases (56).…”

Textbook of Personalized Medicine

“…This approach has potential applications in personal genomics, single-cell genomics and statistical genetics. A method has been described for rapid and cost-effective long-range haplotyping (Kaper et al 2013 ). Genomic DNA is diluted and distributed into multiple aliquots such that each aliquot receives a fraction of a haploid copy.…”