Menu

Auto Tag: Algorithm

June 1, 2021  |  

Effect of coverage depth and haplotype phasing on structural variant detection with PacBio long reads

Each human genome has thousands of structural variants compared to the reference assembly, up to 85% of which are difficult or impossible to detect with Illumina short reads and are only visible with long, multi-kilobase reads. The PacBio RS II and Sequel single molecule, real-time (SMRT) sequencing platforms have made it practical to generate long reads at high throughput. These platforms enable the discovery of structural variants just as short-read platforms did for single nucleotide variants. Numerous software algorithms call structural variants effectively from PacBio long reads, but algorithm sensitivity is lower for insertion variants and all heterozygous variants. Furthermore, the impact of coverage depth and read lengths on sensitivity is not fully characterized. To quantify how zygosity, coverage depth, and read lengths impact the sensitivity of structural variant detection, we obtained high coverage PacBio sequences for three human samples: haploid CHM1, diploid NA12878, and diploid SK-BR-3. For each dataset, reads were randomly subsampled to titrate coverage from 0.5- to 50-fold. The structural variants detected at each coverage were compared to the set at “full” 50-fold coverage. For the diploid samples, additional titrations were performed with reads first partitioned by phase using single nucleotide variants for essentially haploid structural variant discovery. Even at low coverages (1- to 5-fold), PacBio long reads reveal hundreds of structural variants that are not seen in deep 50-fold Illumina whole genome sequences. At moderate 10-fold PacBio coverage, a majority of structural variants are detected. Sensitivity begins to level off at around 40-fold coverage, though it does not fully saturate before 50-fold. Phasing improves sensitivity for all variant types, especially at moderate 10- to 20-fold coverage. Long reads are an effective tool to identify and phase structural variants in the human genome. The majority of variants are detected at moderate 10-fold coverage, and even extremely low long-read coverage (1- to 5-fold) reveals variants that are invisible to short-read sequencing. Performance will continue to improve with better software and longer reads, which will empower studies to connect structural variants to healthy and disease traits in the human population.

Read more
June 1, 2021  |  

Using the PacBio Sequel System to taxonomically and functionally classify metagenomic samples in a trial of patients undergoing fecal microbiota transplantation

Whole-sample shotgun sequencing can provide a more detailed view of a metagenomic community than 16S sequencing, but its use in multi-sample experiments is limited by throughput, cost and analysis complexity. While short-read sequencing technologies offer higher throughput, read lengthss less fewer than 500 bp will rarely cover a gene of interest, and necessitate assembly before further analysis. Assembling large fragments requires sampling each community member at a high depth, significantly increasing the amount of sequencing needed, and limiting the analysis of rare community members. Assembly methods also risk It is also possible to incorrectly combine combining sequences from different community members.

Read more
June 1, 2021  |  

Structural variant detection with low-coverage Pacbio sequencing

Despite amazing progress over the past quarter century in the technology to detect genetic variants, intermediate-sized structural variants (50 bp to 50 kb) have remained difficult to identify. Such variants are too small to detect with array comparative genomic hybridization, but too large to reliably discover with short-read DNA sequencing. Recent de novo assemblies of human genomes have demonstrated the power of PacBio Single Molecule, Real-Time (SMRT) Sequencing to fill this technology gap and sensitively identify structural variants in the human genome. While de novo assembly is the ideal method to identify variants in a genome, it requires high depth of coverage. A structural variant discovery approach that utilizes lower coverage would facilitate evaluation of large patient and population cohorts. Here we introduce such an approach and apply it to 10-fold coverage of several human genomes generated on the PacBio Sequel System. To identify structural variants in low-fold coverage whole genome sequencing data, we apply a reference-based, re-sequencing workflow. First, reads are mapped to the human reference genome with a local aligner. The local alignments often end at structural variant loci. To connect co-linear local alignments across structural variants, we apply a novel algorithm that merges alignments into “chains” and refines the alignment edges. Then, the chained alignments are scanned for windows with an excess of insertions or deletions to identify candidate structural variant loci. Finally, the read support at each putative variant locus is evaluated to produce a variant call. Single nucleotide information is incorporated to phase and evaluate the zygosity of each structural variant. In 10-fold coverage human genome sequence, we identify the vast majority of the structural variants found by de novo assembly, thus demonstrating the power of low-fold coverage SMRT Sequencing to affordably and effectively detect structural variants.

Read more
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.

Read more
June 1, 2021  |  

Full-length transcript profiling with the Iso-Seq method for improved genome annotations

Incomplete annotation of genomes represents a major impediment to understanding biological processes, functional differences between species, and evolutionary mechanisms. Often, genes that are large, embedded within duplicated genomic regions, or associated with repeats are difficult to study by short-read expression profiling and assembly. In addition, most genes in eukaryotic organisms produce alternatively spliced isoforms, broadening the diversity of proteins encoded by the genome, which are difficult to resolve with short-read methods. Short-read RNA sequencing (RNA-seq) works by physically shearing transcript isoforms into smaller pieces and bioinformatically reassembling them, leaving opportunity for misassembly or incomplete capture of the full diversity of isoforms from genes of interest. In contrast, Single Molecule, Real-Time (SMRT) Sequencing directly sequences full-length transcripts without the need for assembly and imputation. Here we apply the Iso-Seq method (long-read RNA sequencing) to detect full-length isoforms and the new IsoPhase algorithm to retrieve allele-specific isoform information for two avian models of vocal learning, Anna’s hummingbird (Calypte anna) and zebra finch (Taeniopygia guttata).

Read more
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.

Read more
June 1, 2021  |  

Haplotyping using full-length transcript sequencing reveals allele-specific expression

An important need in analyzing complex genomes is the ability to separate and phase haplotypes. While whole genome assembly can deliver this information, it cannot reveal whether there is allele-specific gene or isoform expression. The PacBio Iso-Seq method, which can produce high-quality transcript sequences of 10 kb and longer, has been used to annotate many important plant and animal genomes. We present an algorithm called IsoPhase that post-processes Iso-Seq data for transcript-based haplotyping. We applied IsoPhase to a maize Iso-Seq dataset consisting of two homozygous parents and two F1 cross hybrids. We validated the majority of the SNPs called with IsoPhase against matching short read data and identified cases of allele-specific, gene-level and isoform-level expression.

Read more
June 1, 2021  |  

Single molecule high-fidelity (HiFi) Sequencing with >10 kb libraries

Recent improvements in sequencing chemistry and instrument performance combine to create a new PacBio data type, Single Molecule High-Fidelity reads (HiFi reads). Increased read length and improvement in library construction enables average read lengths of 10-20 kb with average sequence identity greater than 99% from raw single molecule reads. The resulting reads have the accuracy comparable to short read NGS but with 50-100 times longer read length. Here we benchmark the performance of this data type by sequencing and genotyping the Genome in a Bottle (GIAB) HG0002 human reference sample from the National Institute of Standards and Technology (NIST). We further demonstrate the general utility of HiFi reads by analyzing multiple clones of Cabernet Sauvignon. Three different clones were sequenced and de novo assembled with the CANU assembly algorithm, generating draft assemblies of very high contiguity equal to or better than earlier assembly efforts using PacBio long reads. Using the Cabernet Sauvignon Clone 8 assembly as a reference, we mapped the HiFi reads generated from Clone 6 and Clone 47 to identify single nucleotide polymorphisms (SNPs) and structural variants (SVs) that are specific to each of the three samples.

Read more
June 1, 2021  |  

Comprehensive variant detection in a human genome with highly accurate long reads

Introduction: Long-read sequencing has been applied successfully to assemble genomes and detect structural variants. However, due to high raw-read error rates (10-15%), it has remained difficult to call small variants from long reads. Recent improvements in library preparation and sequencing chemistry have increased length, accuracy, and throughput of PacBio circular consensus sequencing (CCS) reads, resulting in 10-20kb reads with average read quality above 99%. Materials and Methods: We sequenced a 12kb library from human reference sample HG002 to 18-fold coverage on the PacBio Sequel II System with three SMRT Cells 8M. The CCS algorithm was used to generate highly-accurate (average 99.8%) 11.4kb reads, which were mapped to the hg19 reference with pbmm2. We detected small variants using Google DeepVariant with a model trained for CCS and phased the variants using WhatsHap. Structural variants were detected with pbsv. Variant calls were evaluated against Genome in a Bottle (GIAB) benchmarks. Results: With these reads, DeepVariant achieves SNP and Indel F1 scores of 99.82% and 96.70% against the GIAB truth set, and pbsv achieves 95.94% recall on structural variants longer than 50bp. Using WhatsHap, small variants were phased into haplotype blocks with 105kb N50. The improved mappability of long reads allows us to align to and detect variants in medically relevant genes such as CYP2D6 and PMS2 that have proven “difficult-to-map” with short reads. Conclusions: These highly-accurate long reads combine the mappability and ability to detect structural variants of long reads with the accuracy and ability to detect small variants of short reads.

Read more
June 1, 2021  |  

Unbiased characterization of metagenome composition and function using HiFi sequencing on the PacBio Sequel II System

Recent work comparing metagenomic sequencing methods indicates that a comprehensive picture of the taxonomic and functional diversity of complex communities will be difficult to achieve with short-read technology alone. While the lower cost of short reads has enabled greater sequencing depth, the greater contiguity of long-read assemblies and lack of GC bias in SMRT Sequencing has enabled better gene finding. However, since long-read assembly requires high coverage for error correction, the benefits of unbiased coverage have in the past been lost for low abundance species. SMRT Sequencing performance improvements and the introduction of the Sequel II System has enabled a new, high throughput data type uniquely suited to metagenome characterization: HiFi reads. HiFi reads combine high accuracy with read lengths up to 15 kb, eliminating the need for assembly for most microbiome applications, including functional profiling, gene discovery, and metabolic pathway reconstruction. Here we present the application of the HiFi data type to enable a new method of analyzing metagenomes that does not require assembly.

Read more
June 1, 2021  |  

Comprehensive structural and copy-number variant detection with long reads

To comprehensively detect large variants in human genomes, we have extended pbsv – a structural variant caller for long reads – to call copy-number variants (CNVs) from read-clipping and read-depth signatures. In human germline benchmark samples, we detect more than 300 CNVs spanning around 10 Mb, and we call hundreds of additional events in re-arranged cancer samples. Long-read sequencing of diverse humans has revealed more than 20,000 insertion, deletion, and inversion structural variants spanning more than 12 Mb in a typical human genome. Most of these variants are too large to detect with short reads and too small for array comparative genome hybridization (aCGH). While the standard approaches to calling structural variants with long reads thrive in the 50 bp to 10 kb size range, they tend to miss exactly the large (>50 kb) copy-number variants that are called more readily with aCGH and short reads. Standard algorithms rely on reference-based mapping of reads that fully span a variant or on de novo assembly; and copy-number variants are often too large to be spanned by a single read and frequently involve segmentally duplicated sequence that is not yet included in most de novo assemblies.

Read more
June 1, 2021  |  

Detection and phasing of small variants in Genome in a Bottle samples with highly accurate long reads

Introduction: Long-read PacBio SMRT Sequencing has been applied successfully to assemble genomes and detect structural variants. However, due to high raw read error rates of 10-15%, it has remained difficult to call small variants from long reads. Recent improvements in library preparation, sequencing chemistry, and instrument yield have increased length, accuracy, and throughput of PacBio Circular Consensus (CCS) reads, resulting in 10-20 kb “HiFi” reads with mean read quality above 99%. Materials and Methods: We sequenced 11 kb size-selected libraries from the Genome in a Bottle (GIAB) human reference samples HG001, HG002, and HG005 to approximately 30-fold coverage on the Sequel II System with six SMRT Cells 8M each. The CCS algorithm was used to generate highly accurate (average 99.8%) reads of mean length 10-11 kb, which were then mapped to the hs37d5 reference with pbmm2. We detected small variants using Google DeepVariant and compared these variant calls to GIAB benchmarks. Small variants were then phased with WhatsHap. Results: With these long, highly accurate CCS reads, DeepVariant achieves high SNP and Indel accuracy against the GIAB benchmark truth set for all three reference samples. Using WhatsHap, small variants were phased into haplotype blocks with N50 from 82 to 146 kb. The improved mappability of long reads allows detection of variants in many medically relevant genes such as CYP2D6and PMS2that have proven ‘difficult-to-map’ with short reads. We show that small variant precision and recall remain high down to 15-fold coverage. Conclusions: These highly accurate long reads combine the mappability of noisy long reads with the accuracy and small variant detection utility of short reads, which will allow the detection and phasing of variants in regions that have proven recalcitrant to short read sequencing and variant detection.

Read more
June 1, 2021  |  

Unbiased characterization of metagenome composition and function using HiFi sequencing on the PacBio Sequel II System

Recent work comparing metagenomic sequencing methods indicates that a comprehensive picture of the taxonomic and functional diversity of complex communities will be difficult to achieve with one sequencing technology alone. While the lower cost of short reads has enabled greater sequencing depth, the greater contiguity of long-read assemblies and lack of GC bias in SMRT Sequencing has enabled better gene finding. However, since long-read assembly typically requires high coverage for error correction, these benefits have in the past been lost for low-abundance species. The introduction of the Sequel II System has enabled a new, higher throughput, assembly-optional data type that addresses these challenges: HiFi reads. HiFi reads combine QV20 accuracy with long read lengths, eliminating the need for assembly for most metagenome applications, including gene discovery and metabolic pathway reconstruction. In fact, the read lengths and accuracy of HiFi data match or outperform the quality metrics of most metagenome assemblies, enabling cost-effective recovery of intact genes and operons while omitting the resource intensive and data-inefficient assembly step. Here we present the application of HiFi sequencing to both mock and human fecal samples using full-length 16S and shotgun methods. This proof-of-concept work demonstrates the unique strengths of the HiFi method. First, the high correspondence between the expected community composition,16S and shotgun profiling data reflects low context bias. In addition, every HiFi read yields ~5-8 predicted genes, without assembly, using standard tools. If assembly is desired, excellent results can be achieved with Canu and contig binning tools. In summary, HiFi sequencing is a new, cost-effective option for high-resolution functional profiling of metagenomes which complements existing short read workflows.

Read more
June 1, 2021  |  

Copy-number variant detection with PacBio long reads

Long-read sequencing of diverse humans has revealed more than 20,000 insertion, deletion, and inversion structural variants spanning more than 12 Mb in a healthy human genome. Most of these variants are too large to detect with short reads and too small for array comparative genome hybridization (aCGH). While the standard approaches to calling structural variants with long reads thrive in the 50 bp to 10 kb size range, they tend to miss exactly the large (>50 kb) copy-number variants that are called more readily with aCGH. Standard algorithms rely on reference-based mapping of reads that fully span a variant or on de novo assembly; and copy-number variants are often too large to be spanned by a single read and frequently involve segmentally duplicated sequence that is not yet included in most de novo assemblies. To comprehensively detect large variants in human genomes, we extended pbsv – a structural variant caller for long reads – to call copy-number variants (CNVs) from read-clipping and read-depth signatures. In human germline benchmark samples, we detect more than 300 CNVs spanning around 10 Mb, and we call hundreds of additional events in re-arranged cancer samples. Together with insertion, deletion, inversion, duplication, and translocation calling from spanning reads, this allows pbsv to comprehensively detect large variants from a single data type.

Read more
June 1, 2021  |  

Metagenomic analysis of type II diabetes gut microbiota using PacBio HiFi reads reveals taxonomic and functional differences

In the past decade, the human microbiome has been increasingly shown to play a major role in health. For example, imbalances in gut microbiota appear to be associated with Type II diabetes mellitus (T2DM) and cardiovascular disease. Coronary artery disease (CAD) is a major determinant of the long-term prognosis among T2DM patients, with a 2- to 4-fold increased mortality risk when present. However, the exact microbial strains or functions implicated in disease need further investigation. From a large study with 523 participants (185 healthy controls, 186 T2DM patients without CAD, and 106 T2DM patients with CAD), 3 samples from each patient group were selected for long read sequencing. Each sample was prepared and sequenced on one Sequel II System SMRT Cell, to assess whether long accurate PacBio HiFi reads could yield additional insights to those made using short reads. Each of the 9 samples was subject to metagenomic assembly and binning, taxonomic classification and functional profiling. Results from metagenomic assembly and binning show that it is possible to generate a significant number of complete MAGs (Metagenome Assembled Genomes) from each sample, with over half of the high-quality MAGs being represented by a single circular contig. We show that differences found in taxonomic and functional profiles of healthy versus diabetic patients in the small 9-sample study align with the results of the larger study, as well as with results reported in literature. For example, the abundances of beneficial short- chain fatty acid (SCFA) producers such as Phascolarctobacterium faecium and Faecalibacterium prausnitzii were decreased in T2DM gut microbiota in both studies, while the abundances of quinol and quinone biosynthesis pathways were increased as compared to healthy controls. In conclusion, metagenomic analysis of long accurate HiFi reads revealed important taxonomic and functional differences in T2DM versus healthy gut microbiota. Furthermore, metagenome assembly of long HiFi reads led to the recovery of many complete MAGs and a significant number of complete circular bacterial chromosome sequences.

Read more

Subscribe for blog updates:

Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.