Accurate whole-genome sequencing and haplotyping from 10 to 20 human cells

Abstract

Recent advances in whole genome sequencing have brought the vision of personal genomics and genomic medicine closer to reality. However, current methods lack clinical accuracy and the ability to describe the context (haplotypes) in which genome variants co-occur in a cost-effective manner. Here we describe a low-cost DNA sequencing and haplotyping process, Long Fragment Read (LFR) technology, similar to sequencing long single DNA molecules without cloning or separation of metaphase chromosomes. In this study, ten LFR libraries were made using only ~100 pg of human DNA per sample. Up to 97% of the heterozygous single nucleotide variants (SNVs) were assembled into long haplotype contigs. Removal of false positive SNVs not phased by multiple LFR haplotypes resulted in a final genome error rate of 1 in 10 Mb. Cost-effective and accurate genome sequencing and haplotyping from 10-20 human cells, as demonstrated here, will enable comprehensive genetic studies and diverse clinical applications.

Whole-genome haplotyping by dilution, amplification, and sequencing

“…Doubling the number of dilution aliquots from 96 to 192 increased the average haplotype block size 3.7-or 1.6-fold for the YRI and CEPH genomes, respectively, and decreased the number of haplotype blocks 3.4-or 1.5-fold, respectively (Table S1 and Table 1). These haplotyping metrics can surely be improved upon by increasing the number of dilution aliquots or by using higher-molecular-weight DNA template molecules that are more likely to span SNP deserts (20) (SI Text, section S6 and Table S6). Furthermore, most of the data analysis was performed with publicly available algorithms that were not custom-designed for these experiments.…”

“…However, a significant number of prescreen PCR reactions are required as each aliquot needs to be cross-referenced against all regions of interest, thereby limiting the number of target regions that can be addressed. A recently published study overcomes these limitations by combining the dilution-amplification method with massively parallel sequencing using Complete Genomics' service for whole-genome haplotyping (20).…”

Sequencing of isolated sperm cells for direct haplotyping of a human genome

“…Sequencing of paired-end reads (Levy et al 2007;McKernan et al 2009) or sequencing of long DNA fragments (Kitzman et al 2011;Suk et al 2011;Peters et al 2012) have been used to link multiple variant loci into large haplotype blocks (N50 values of up to 1.0 Mb), although none of these blocks span entire chromosomes. Other approaches involve the physical separation of chromosomes and include the use of somatic cell hybrids (Douglas et al 2001), polony sequencing (Zhang et al 2006), chromosome microdissection (Ma et al 2010) or chromosome sorting by fluorescenceactivated cell sorting (FACS) or microfluidic manipulation (Fan et al 2011;Yang et al 2011).…”

Human Genetics and Clinical Aspects of Neurodevelopmental Disorders

“…The same is true for genotyping microarrays, which are becoming increasingly denser with various markers while maintaining a relatively stable cost [187]. With rapid advancements in sequencing technologies [188] and improved haplotype-phasing [189,190], high-throughput sequencing (HTS) data on the genomes of a diverse number of species are being generated at an unprecedented rate. The development of bioinformatics tools for handling these data has been somewhat lagged in response, creating a gap between the massive data being generated, and the ability to fully exploit the biological content of these data.…”