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Auto Tag: Diploid assembly

June 1, 2021

Haplotyping of full-length transcript reads from long-read sequencing can reveal allelic imbalances in isoform expression

The Pacific Biosciences Iso-Seq method, which can produce high-quality isoform sequences of 10 kb and longer, has been used to annotate many important plant and animal genomes. Here, we develop an algorithm called IsoPhase that postprocesses Iso-Seq data to retrieve allele specific isoform information. Using simulated data, we show that for both diploid and tetraploid genomes, IsoPhase results in good SNP recovery with low FDR at error rates consistent with CCS reads. We apply IsoPhase to a haplotyperesolved genome assembly and multiple fetal tissue Iso-Seq dataset from a F1 cross of Angus x Brahman cattle subspecies. IsoPhase-called haplotypes were validated by the phased assembly and demonstrate the potential for revealing allelic imbalances in isoform expression.

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June 1, 2021

Best practices for whole genome sequencing using the Sequel System

Plant and animal whole genome sequencing has proven to be challenging, particularly due to genome size, high density of repetitive elements and heterozygosity. The Sequel System delivers long reads, high consensus accuracy and uniform coverage, enabling more complete, accurate, and contiguous assemblies of these large complex genomes. The latest Sequel chemistry increases yield up to 8 Gb per SMRT Cell for long insert libraries >20 kb and up to 10 Gb per SMRT Cell for libraries >40 kb. In addition, the recently released SMRTbell Express Template Prep Kit reduces the time (~3 hours) and DNA input (~3 µg), making the workflow easy to use for multi- SMRT Cell projects. Here, we recommend the best practices for whole genome sequencing and de novo assembly of complex plant and animal genomes. Guidelines for constructing large-insert SMRTbell libraries (>30 kb) to generate optimal read lengths and yields using the latest Sequel chemistry are presented. We also describe ways to maximize library yield per preparation from as littles as 3 µg of sheared genomic DNA. The combination of these advances makes plant and animal whole genome sequencing a practical application of the Sequel System.

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June 1, 2021

FALCON-Phase integrates PacBio and HiC data for de novo assembly, scaffolding and phasing of a diploid Puerto Rican genome (HG00733)

Haplotype-resolved genomes are important for understanding how combinations of variants impact phenotypes. The study of disease, quantitative traits, forensics, and organ donor matching are aided by phased genomes. Phase is commonly resolved using familial data, population-based imputation, or by isolating and sequencing single haplotypes using fosmids, BACs, or haploid tissues. Because these methods can be prohibitively expensive, or samples may not be available, alternative approaches are required. de novo genome assembly with PacBio Single Molecule, Real-Time (SMRT) data produces highly contiguous, accurate assemblies. For non-inbred samples, including humans, the separate resolution of haplotypes results in higher base accuracy and more contiguous assembled sequences. Two primary methods exist for phased diploid genome assembly. The first, TrioCanu requires Illumina data from parents and PacBio data from the offspring. The long reads from the child are partitioned into maternal and paternal bins using parent-specific sequences; the separate PacBio read bins are then assembled, generating two fully phased genomes. An alternative approach (FALCON-Unzip) does not require parental information and separates PacBio reads, during genome assembly, using heterozygous SNPs. The length of haplotype phase blocks in FALCON-Unzip is limited by the magnitude and distribution of heterozygosity, the length of sequence reads, and read coverage. Because of this, FALCON-Unzip contigs typically contain haplotype-switch errors between phase blocks, resulting in primary contig of mixed parental origin. We developed FALCON-Phase, which integrates Hi-C data downstream of FALCON-Unzip to resolve phase switches along contigs. We applied the method to a human (Puerto Rican, HG00733) and non-human genome assemblies and evaluated accuracy using samples with trio data. In a cattle genome, we observe >96% accuracy in phasing when compared to TrioCanu assemblies as well as parental SNPs. For a high-quality PacBio assembly (>90-fold Sequel coverage) of a Puerto Rican individual we scaffolded the FALCON-Phase contigs, and re-phased the contigs creating a de novo scaffolded, phased diploid assembly with chromosome-scale contiguity.

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April 21, 2020

Chromosome-length haplotigs for yak and cattle from trio binning assembly of an F1 hybrid

Background Assemblies of diploid genomes are generally unphased, pseudo-haploid representations that do not correctly reconstruct the two parental haplotypes present in the individual sequenced. Instead, the assembly alternates between parental haplotypes and may contain duplications in regions where the parental haplotypes are sufficiently different. Trio binning is an approach to genome assembly that uses short reads from both parents to classify long reads from the offspring according to maternal or paternal haplotype origin, and is thus helped rather than impeded by heterozygosity. Using this approach, it is possible to derive two assemblies from an individual, accurately representing both parental contributions in their entirety with higher continuity and accuracy than is possible with other methods.Results We used trio binning to assemble reference genomes for two species from a single individual using an interspecies cross of yak (Bos grunniens) and cattle (Bos taurus). The high heterozygosity inherent to interspecies hybrids allowed us to confidently assign >99% of long reads from the F1 offspring to parental bins using unique k-mers from parental short reads. Both the maternal (yak) and paternal (cattle) assemblies contain over one third of the acrocentric chromosomes, including the two largest chromosomes, in single haplotigs.Conclusions These haplotigs are the first vertebrate chromosome arms to be assembled gap-free and fully phased, and the first time assemblies for two species have been created from a single individual. Both assemblies are the most continuous currently available for non-model vertebrates.MbmegabaseskbkilobasesMYAmillions of years agoMHCmajor histocompatibility complexSMRTsingle molecule real time

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April 21, 2020

A robust benchmark for germline structural variant detection

New technologies and analysis methods are enabling genomic structural variants (SVs) to be detected with ever-increasing accuracy, resolution, and comprehensiveness. Translating these methods to routine research and clinical practice requires robust benchmark sets. We developed the first benchmark set for identification of both false negative and false positive germline SVs, which complements recent efforts emphasizing increasingly comprehensive characterization of SVs. To create this benchmark for a broadly consented son in a Personal Genome Project trio with broadly available cells and DNA, the Genome in a Bottle (GIAB) Consortium integrated 19 sequence-resolved variant calling methods, both alignment- and de novo assembly-based, from short-, linked-, and long-read sequencing, as well as optical and electronic mapping. The final benchmark set contains 12745 isolated, sequence-resolved insertion and deletion calls =50 base pairs (bp) discovered by at least 2 technologies or 5 callsets, genotyped as heterozygous or homozygous variants by long reads. The Tier 1 benchmark regions, for which any extra calls are putative false positives, cover 2.66 Gbp and 9641 SVs supported by at least one diploid assembly. Support for SVs was assessed using svviz with short-, linked-, and long-read sequence data. In general, there was strong support from multiple technologies for the benchmark SVs, with 90 % of the Tier 1 SVs having support in reads from more than one technology. The Mendelian genotype error rate was 0.3 %, and genotype concordance with manual curation was >98.7 %. We demonstrate the utility of the benchmark set by showing it reliably identifies both false negatives and false positives in high-quality SV callsets from short-, linked-, and long-read sequencing and optical mapping.

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April 21, 2020

Extended haplotype phasing of de novo genome assemblies with FALCON-Phase

Haplotype-resolved genome assemblies are important for understanding how combinations of variants impact phenotypes. These assemblies can be created in various ways, such as use of tissues that contain single-haplotype (haploid) genomes, or by co-sequencing of parental genomes, but these approaches can be impractical in many situations. We present FALCON-Phase, which integrates long-read sequencing data and ultra-long-range Hi-C chromatin interaction data of a diploid individual to create high-quality, phased diploid genome assemblies. The method was evaluated by application to three datasets, including human, cattle, and zebra finch, for which high-quality, fully haplotype resolved assemblies were available for benchmarking. Phasing algorithm accuracy was affected by heterozygosity of the individual sequenced, with higher accuracy for cattle and zebra finch (>97%) compared to human (82%). In addition, scaffolding with the same Hi-C chromatin contact data resulted in phased chromosome-scale scaffolds.

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April 21, 2020

Chromosome-scale assemblies reveal the structural evolution of African cichlid genomes.

African cichlid fishes are well known for their rapid radiations and are a model system for studying evolutionary processes. Here we compare multiple, high-quality, chromosome-scale genome assemblies to elucidate the genetic mechanisms underlying cichlid diversification and study how genome structure evolves in rapidly radiating lineages.We re-anchored our recent assembly of the Nile tilapia (Oreochromis niloticus) genome using a new high-density genetic map. We also developed a new de novo genome assembly of the Lake Malawi cichlid, Metriaclima zebra, using high-coverage Pacific Biosciences sequencing, and anchored contigs to linkage groups (LGs) using 4 different genetic maps. These new anchored assemblies allow the first chromosome-scale comparisons of African cichlid genomes. Large intra-chromosomal structural differences (~2-28 megabase pairs) among species are common, while inter-chromosomal differences are rare (<10 megabase pairs total). Placement of the centromeres within the chromosome-scale assemblies identifies large structural differences that explain many of the karyotype differences among species. Structural differences are also associated with unique patterns of recombination on sex chromosomes. Structural differences on LG9, LG11, and LG20 are associated with reduced recombination, indicative of inversions between the rock- and sand-dwelling clades of Lake Malawi cichlids. M. zebra has a larger number of recent transposable element insertions compared with O. niloticus, suggesting that several transposable element families have a higher rate of insertion in the haplochromine cichlid lineage.This study identifies novel structural variation among East African cichlid genomes and provides a new set of genomic resources to support research on the mechanisms driving cichlid adaptation and speciation. © The Author(s) 2019. Published by Oxford University Press.

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