Phasebook: haplotype-aware de novo assembly of diploid genomes from long reads
ABSTRACT: Haplotype-aware diploid genome assembly is crucial in genomics, precision medicine, and many other disciplines. Long-read sequencing technologies have greatly improved genome assembly. However, current long-read assemblers are either reference based, so introduce biases, or fail to capture the haplotype diversity of diploid genomes. We present phasebook, a de novo approach for reconstructing the haplotypes of diploid genomes from long reads. phasebook outperforms other approaches in terms of haplotype coverage by large margins, in addition to achieving competitive performance in terms of assembly errors and assembly contiguity.
The online version contains supplementary material available at (10.1186/s13059-021-02512-x).
Project description:Long-read de novo genome assembly continues to advance rapidly. However, there is a lack of effective tools to accurately evaluate the assembly results, especially for structural errors. We present Inspector, a reference-free long-read de novo assembly evaluator which faithfully reports types of errors and their precise locations. Notably, Inspector can correct the assembly errors based on consensus sequences derived from raw reads covering erroneous regions. Based on in silico and long-read assembly results from multiple long-read data and assemblers, we demonstrate that in addition to providing generic metrics, Inspector can accurately identify both large-scale and small-scale assembly errors. <h4>Supplementary Information</h4> The online version contains supplementary material available at 10.1186/s13059-021-02527-4.
Project description:BACKGROUND:Recent developments in third-gen long read sequencing and diploid-aware assemblers have resulted in the rapid release of numerous reference-quality assemblies for diploid genomes. However, assembly of highly heterozygous genomes is still problematic when regional heterogeneity is so high that haplotype homology is not recognised during assembly. This results in regional duplication rather than consolidation into allelic variants and can cause issues with downstream analysis, for example variant discovery, or haplotype reconstruction using the diploid assembly with unpaired allelic contigs. RESULTS:A new pipeline-Purge Haplotigs-was developed specifically for third-gen sequencing-based assemblies to automate the reassignment of allelic contigs, and to assist in the manual curation of genome assemblies. The pipeline uses a draft haplotype-fused assembly or a diploid assembly, read alignments, and repeat annotations to identify allelic variants in the primary assembly. The pipeline was tested on a simulated dataset and on four recent diploid (phased) de novo assemblies from third-generation long-read sequencing, and compared with a similar tool. After processing with Purge Haplotigs, haploid assemblies were less duplicated with minimal impact on genome completeness, and diploid assemblies had more pairings of allelic contigs. CONCLUSIONS:Purge Haplotigs improves the haploid and diploid representations of third-gen sequencing based genome assemblies by identifying and reassigning allelic contigs. The implementation is fast and scales well with large genomes, and it is less likely to over-purge repetitive or paralogous elements compared to alignment-only based methods. The software is available at https://bitbucket.org/mroachawri/purge_haplotigs under a permissive MIT licence.
Project description:<h4>Summary</h4>De novo assembly is a difficult issue for heterozygous diploid genomes. The advent of high-throughput short-read and long-read sequencing technologies provides both new challenges and potential solutions to the issue. Here, we present HaploMerger2 (HM2), an automated pipeline for rebuilding both haploid sub-assemblies from the polymorphic diploid genome assembly. It is designed to work on pre-existing diploid assemblies, which are typically created by using de novo assemblers. HM2 can process any diploid assemblies, but it is especially suitable for diploid assemblies with high heterozygosity (?3%), which can be difficult for other tools. This pipeline also implements flexible and sensitive assembly error detection, a hierarchical scaffolding procedure and a reliable gap-closing method for haploid sub-assemblies. Using HM2, we demonstrate that two haploid sub-assemblies reconstructed from a real, highly-polymorphic diploid assembly show greatly improved continuity.<h4>Availability and implementation</h4>Source code, executables and the testing dataset are freely available at https://github.com/mapleforest/HaploMerger2/releases/.<h4>Contact</h4>firstname.lastname@example.org.<h4>Supplementary information</h4>Supplementary data are available at Bioinformatics online.
Project description:Single-molecule sequencing technologies have recently been commercialized by Pacific Biosciences and Oxford Nanopore with the promise of sequencing long DNA fragments (kilobases to megabases order) and then, using efficient algorithms, provide high quality assemblies in terms of contiguity and completeness of repetitive regions. However, the error rate of long-read technologies is higher than that of short-read technologies. This has a direct consequence on the base quality of genome assemblies, particularly in coding regions where sequencing errors can disrupt the coding frame of genes. In the case of diploid genomes, the consensus of a given gene can be a mixture between the two haplotypes and can lead to premature stop codons. Several methods have been developed to polish genome assemblies using short reads and generally, they inspect the nucleotide one by one, and provide a correction for each nucleotide of the input assembly. As a result, these algorithms are not able to properly process diploid genomes and they typically switch from one haplotype to another. Herein we proposed Hapo-G (Haplotype-Aware Polishing Of Genomes), a new algorithm capable of incorporating phasing information from high-quality reads (short or long-reads) to polish genome assemblies and in particular assemblies of diploid and heterozygous genomes.
Project description:SUMMARY:While next-generation sequencing (NGS) has dramatically increased the availability of genomic data, phased genome assembly and structural variant (SV) analyses are limited by NGS read lengths. Long-read sequencing from Pacific Biosciences and NGS barcoding from 10x Genomics hold the potential for far more comprehensive views of individual genomes. Here, we present MsPAC, a tool that combines both technologies to partition reads, assemble haplotypes (via existing software) and convert assemblies into high-quality, phased SV predictions. MsPAC represents a framework for haplotype-resolved SV calls that moves one step closer to fully resolved, diploid genomes. AVAILABILITY AND IMPLEMENTATION:https://github.com/oscarlr/MsPAC. SUPPLEMENTARY INFORMATION:Supplementary data are available at Bioinformatics online.
Project description:<h4>Motivation</h4>Achieving a near complete understanding of how the genome of an individual affects the phenotypes of that individual requires deciphering the order of variations along homologous chromosomes in species with diploid genomes. However, true diploid assembly of long-range haplotypes remains challenging.<h4>Results</h4>To address this, we have developed Haplotype-resolved Assembly for Synthetic long reads using a Trio-binning strategy, or HAST, which uses parental information to classify reads into maternal or paternal. Once sorted, these reads are used to independently de novo assemble the parent-specific haplotypes. We applied HAST to co-barcoded second-generation sequencing data from an Asian individual, resulting in a haplotype assembly covering 94.7% of the reference genome with a scaffold N50 longer than 11 Mb. The high haplotyping precision (∼99.7%) and recall (∼95.9%) represents a substantial improvement over the commonly used tool for assembling co-barcoded reads (Supernova), and is comparable to a trio-binning-based third generation long-read based assembly method (TrioCanu) but with a significantly higher single-base accuracy (up to 99.99997% (Q65)). This makes HAST a superior tool for accurate haplotyping and future haplotype-based studies.<h4>Availability</h4>The code of the analysis is available at https://github.com/BGI-Qingdao/HAST.<h4>Supplementary information</h4>Supplementary data are available at Bioinformatics online.
Project description:<h4>Motivation</h4>Rapid development in long-read sequencing and scaffolding technologies is accelerating the production of reference-quality assemblies for large eukaryotic genomes. However, haplotype divergence in regions of high heterozygosity often results in assemblers creating two copies rather than one copy of a region, leading to breaks in contiguity and compromising downstream steps such as gene annotation. Several tools have been developed to resolve this problem. However, they either focus only on removing contained duplicate regions, also known as haplotigs, or fail to use all the relevant information and hence make errors.<h4>Results</h4>Here we present a novel tool, purge_dups, that uses sequence similarity and read depth to automatically identify and remove both haplotigs and heterozygous overlaps. In comparison with current tools, we demonstrate that purge_dups can reduce heterozygous duplication and increase assembly continuity while maintaining completeness of the primary assembly. Moreover, purge_dups is fully automatic and can easily be integrated into assembly pipelines.<h4>Availability and implementation</h4>The source code is written in C and is available at https://github.com/dfguan/purge_dups.<h4>Supplementary information</h4>Supplementary data are available at Bioinformatics online.
Project description:While genome assembly projects have been successful in many haploid and inbred species, the assembly of noninbred or rearranged heterozygous genomes remains a major challenge. To address this challenge, we introduce the open-source FALCON and FALCON-Unzip algorithms (https://github.com/PacificBiosciences/FALCON/) to assemble long-read sequencing data into highly accurate, contiguous, and correctly phased diploid genomes. We generate new reference sequences for heterozygous samples including an F1 hybrid of Arabidopsis thaliana, the widely cultivated Vitis vinifera cv. Cabernet Sauvignon, and the coral fungus Clavicorona pyxidata, samples that have challenged short-read assembly approaches. The FALCON-based assemblies are substantially more contiguous and complete than alternate short- or long-read approaches. The phased diploid assembly enabled the study of haplotype structure and heterozygosities between homologous chromosomes, including the identification of widespread heterozygous structural variation within coding sequences.
Project description:The PacBio® HiFi sequencing method yields highly accurate long-read sequencing datasets with read lengths averaging 10-25?kb and accuracies greater than 99.5%. These accurate long reads can be used to improve results for complex applications such as single nucleotide and structural variant detection, genome assembly, assembly of difficult polyploid or highly repetitive genomes, and assembly of metagenomes. Currently, there is a need for sample data sets to both evaluate the benefits of these long accurate reads as well as for development of bioinformatic tools including genome assemblers, variant callers, and haplotyping algorithms. We present deep coverage HiFi datasets for five complex samples including the two inbred model genomes Mus musculus and Zea mays, as well as two complex genomes, octoploid Fragaria?×?ananassa and the diploid anuran Rana muscosa. Additionally, we release sequence data from a mock metagenome community. The datasets reported here can be used without restriction to develop new algorithms and explore complex genome structure and evolution. Data were generated on the PacBio Sequel II System.
Project description:High-quality chromosome-scale haplotype sequences of diploid genomes, polyploid genomes, and metagenomes provide important insights into genetic variation associated with disease and biodiversity. However, whole-genome short read sequencing does not yield haplotype information spanning whole chromosomes directly. Computational assembly of shorter haplotype fragments is required for haplotype reconstruction, which can be challenging owing to limited fragment lengths and high haplotype and repeat variability across genomes. Recent advancements in long-read and chromosome-scale sequencing technologies, alongside computational innovations, are improving the reconstruction of haplotypes at the level of whole chromosomes. Here, we review recent and discuss methodological progress and perspectives in these areas.