A comprehensively molecular haplotype-resolved genome of a European individual

Abstract

Independent determination of both haplotype sequences of an individual genome is essential to relate genetic variation to genome function, phenotype, and disease. To address the importance of phase, we have generated the most complete haplotype-resolved genome to date, “Max Planck One” (MP1), by fosmid pool-based next generation sequencing. Virtually all SNPs (>99%) and 80,000 indels were phased into haploid sequences of up to 6.3 Mb (N50 ∼1 Mb). The completeness of phasing allowed determination of the concrete molecular haplotype pairs for the vast majority of genes (81%) including potential regulatory sequences, of which >90% were found to be constituted by two different molecular forms. A subset of 159 genes with potentially severe mutations in either cis or trans configurations exemplified in particular the role of phase for gene function, disease, and clinical interpretation of personal genomes (e.g., BRCA1). Extended genomic regions harboring manifold combinations of physically and/or functionally related genes and regulatory elements were resolved into their underlying “haploid landscapes,” which may define the functional genome. Moreover, the majority of genes and functional sequences were found to contain individual or rare SNPs, which cannot be phased from population data alone, emphasizing the importance of molecular phasing for characterizing a genome in its molecular individuality. Our work provides the foundation to understand that the distinction of molecular haplotypes is essential to resolve the (inherently individual) biology of genes, genomes, and disease, establishing a reference point for “phase-sensitive” personal genomics. MP1's annotated haploid genomes are available as a public resource.

De novo assembly and phasing of a Korean human genome

“…Phasing was performed with PacBio long reads, Illumina short reads, 10X Genomics linked reads 4 (30× ), and reads from BACs representing a single haplotype (47× ). Heterozygous SNVs called from these methods are unambiguously assigned to two alternative phases, producing phased blocks with an N50 length of 11.6 Mb, considerably longer than previously reported 4,6,8,15,16 (Table 1). We assessed the accuracy of the phased blocks against the end sequences of BACs, and found a long-range switch error rate to be under 0.3%.…”

The oyster genome reveals stress adaptation and complexity of shell formation

“…1 Tables 5 and 6). Fosmid pooling has been used for re-sequencing 16,17 , and our results show that the combination of fosmid pooling, NGS and hierarchical assembly provides a new, cost-effective alternative for de novo sequencing and assembly of complex genomes.…”

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

“…Given that phasing can be uncertain in some regions of segmental duplication (see above), it should be noted that 11 SNPs, found within four of the 17 genes (PDE4DIP, HYDIN, PCDHB7, FAM175A) fall within known segmental duplications, and the existence of compound heterozygosity within these genes remains questionable at present. The subject of this study has been generally healthy; and although the current analysis suggests that he may carry 10-20 genes that encode only defective protein products, there is increasing evidence that this could be a common feature of human genomes (Suk et al 2011;MacArthur et al 2012). …”

“…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).…”