Menu

Asset Tag: Circular consensus sequencing

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

Amplification-free targeted enrichment and SMRT Sequencing of repeat-expansion genomic regions

Targeted sequencing has proven to be an economical means of obtaining sequence information for one or more defined regions of a larger genome. However, most target enrichment methods are reliant upon some form of amplification. Amplification removes the epigenetic marks present in native DNA, and some genomic regions, such as those with extreme GC content and repetitive sequences, are recalcitrant to faithful amplification. Yet, a large number of genetic disorders are caused by expansions of repeat sequences. Furthermore, for some disorders, methylation status has been shown to be a key factor in the mechanism of disease.

Read more
June 1, 2021

Allelic specificity of immunoglobulin heavy chain (IGH@) translocation in B-cell acute lymphoblastic leukemia (B-ALL) unveiled by long-read sequencing

Oncogenic fusion of IGH-DUX4 has recently been reported as a hallmark that defines a B-ALL subtype present in up to 7% of adolescents and young adults B-ALL. The translocation of DUX4 into IGH results in aberrant activation of DUX4 by hijacking the intronic IGH enhancer (Eµ). How IGH-DUX4 translocation interplays with IGH allelic exclusion was never been explored. We investigated this in Nalm6 B-ALL cell line, using long-read (PacBio Iso-Seq method and 10X Chromium WGS), short-read (Illumina total stranded RNA and WGS), epigenome (H3K27ac ChIP-seq, ATAC-seq) and 3-D genome (Hi-C, H3K27ac HiChIP, Capture-C).

Read more
June 1, 2021

High-throughput SMRT Sequencing of clinically relevant targets

Targeted sequencing with Sanger as well as short read based high throughput sequencing methods is standard practice in clinical genetic testing. However, many applications beyond SNP detection have remained somewhat obstructed due to technological challenges. With the advent of long reads and high consensus accuracy, SMRT Sequencing overcomes many of the technical hurdles faced by Sanger and NGS approaches, opening a broad range of untapped clinical sequencing opportunities. Flexible multiplexing options, highly adaptable sample preparation method and newly improved two well-developed analysis methods that generate highly-accurate sequencing results, make SMRT Sequencing an adept method for clinical grade targeted sequencing. The Circular Consensus Sequencing (CCS) analysis pipeline produces QV 30 data from each single intra-molecular multi-pass polymerase read, making it a reliable solution for detecting minor variant alleles with frequencies as low as 1 %. Long Amplicon Analysis (LAA) makes use of insert spanning full-length subreads originating from multiple individual copies of the target to generate highly accurate and phased consensus sequences (>QV50), offering a unique advantage for imputation free allele segregation and haplotype phasing. Here we present workflows and results for a range of SMRT Sequencing clinical applications. Specifically, we illustrate how the flexible multiplexing options, simple sample preparation methods and new developments in data analysis tools offered by PacBio in support of Sequel System 5.1 can come together in a variety of experimental designs to enable applications as diverse as high throughput HLA typing, mitochondrial DNA sequencing and viral vector integrity profiling of recombinant adeno-associated viral genomes (rAAV).

Read more
June 1, 2021

Amplification-free, CRISPR-Cas9 targeted enrichment and SMRT Sequencing of repeat-expansion disease causative genomic regions

Targeted sequencing has proven to be economical for obtaining sequence information for defined regions of the genome. However, most target enrichment methods are reliant upon some form of amplification which can negatively impact downstream analysis. For example, amplification removes epigenetic marks present in native DNA, including nucleotide methylation, which are hypothesized to contribute to disease mechanisms in some disorders. In addition, some genomic regions known to be causative of many genetic disorders have extreme GC content and/or repetitive sequences that tend to be recalcitrant to faithful amplification. We have developed a novel, amplification-free enrichment technique that employs the CRISPR/Cas9 system to target individual genes. This method, in conjunction with the long reads, high consensus accuracy, and uniform coverage of SMRT Sequencing, allows accurate sequence analysis of complex genomic regions that cannot be investigated with other technologies. Using this strategy, we have successfully targeted a number of repeat expansion disorder loci (HTT, FMR1, ATXN10, C9orf72).With this data, we demonstrate the ability to isolate thousands of individual on-target molecules and, using the Sequel System, accurately sequence through long repeats regardless of the extreme GC-content. The method is compatible with multiplexing of multiple target loci and multiple samples in a single reaction. Furthermore, because there is no amplification step, this technique also preserves native DNA molecules for sequencing, allowing for the direct detection and characterization of epigenetic signatures. To this end, we demonstrate the detection of 5-mC in the CGG repeat of the FMR1 gene that is responsible for Fragile X syndrome.

Read more
June 1, 2021

A simple segue from Sanger to high-throughput SMRT Sequencing with a M13 barcoding system

High-throughput NGS methods are increasingly utilized in the clinical genomics market. However, short-read sequencing data continues to remain challenged by mapping inaccuracies in low complexity regions or regions of high homology and may not provide adequate coverage within GC-rich regions of the genome. Thus, the use of Sanger sequencing remains popular in many clinical sequencing labs as the gold standard approach for orthogonal validation of variants and to interrogate missed regions poorly covered by second-generation sequencing. The use of Sanger sequencing can be less than ideal, as it can be costly for high volume assays and projects. Additionally, Sanger sequencing generates read lengths shorter than the region of interest, which limits its ability to accurately phase allelic variants. High-throughput SMRT Sequencing overcomes the challenges of both the first- and second-generation sequencing methods. PacBio’s long read capability allows sequencing of full-length amplicons

Read more
June 1, 2021

Comprehensive variant detection in a human genome with PacBio high-fidelity reads

Human genomic variations range in size from single nucleotide substitutions to large chromosomal rearrangements. Sequencing technologies tend to be optimized for detecting particular variant types and sizes. Short reads excel at detecting SNVs and small indels, while long or linked reads are typically used to detect larger structural variants or phase distant loci. Long reads are more easily mapped to repetitive regions, but tend to have lower per-base accuracy, making it difficult to call short variants. The PacBio Sequel System produces two main data types: long continuous reads (up to 100 kbp), generated by single passes over a long template, and Circular Consensus Sequence (CCS) reads, generated by calculating the consensus of many sequencing passes over a single shorter template (500 bp to 20 kbp). The long-range information in continuous reads is useful for genome assembly and structural variant detection. The higher base accuracy of CCS effectively detects and phases short variants in single molecules. Recent improvements in library preparation protocols and sequencing chemistry have increased the length, accuracy, and throughput of CCS reads. For the human sample HG002, we collected 28-fold coverage 15 kbp high-fidelity CCS reads with an average read quality above Q20 (99% accuracy). The length and accuracy of these reads allow us to detect SNVs, indels, and structural variants not only in the Genome in a Bottle (GIAB) high confidence regions, but also in segmental duplications, HLA loci, and clinically relevant “difficult-to-map” genes. As with continuous long reads, we call structural variants at 90.0% recall compared to the GIAB structural variant benchmark “truth” set, with the added advantages of base pair resolution for variant calls and improved recall at compound heterozygous loci. With minimap2 alignments, GATK4 HaplotypeCaller variant calls, and simple variant filtration, we have achieved a SNP F-Score of 99.51% and an INDEL F-Score of 80.10% against the GIAB short variant benchmark “truth” set, in addition to calling variants outside of the high confidence region established by GIAB using previous technologies. With the long-range information available in 15 kbp reads, we applied the read-backed phasing tool WhatsHap to generate phase blocks with a mean length of 65 kbp across the entire genome. Using an alignment-based approach, we typed all major MHC class I and class II genes to at least 3-field precision. This new data type has the potential to expand the GIAB high confidence regions and “truth” benchmark sets to many previously difficult-to-map genes and allow a single sequencing protocol to address both short variants and large structural variants.

Read more
June 1, 2021

Library prep and bioinformatics improvements for full-length transcript sequencing on the PacBio Sequel System

The PacBio Iso-Seq method produces high-quality, full-length transcripts of up to 10 kb and longer and has been used to annotate many important plant and animal genomes. Here we describe an improved, simplified library workflow and analysis pipeline that reduces library preparation time, RNA input, and cost. The Iso-Seq V2 Express workflow is a one day protocol that requires only ~300 ng of total RNA input while also reducing the number of reverse transcription and amplification steps down to single reactions. Compared with the previous workflow, the Iso-Seq V2 Express workflow increases the percentage of full-length (FL) reads while achieving a higher average transcript length. At the same time, the Iso-Seq 3 analysis recently released in the SMRT Link 6.0 software is a major improvement over previous versions. Iso-Seq 3 is highly accurate at detecting and removing library artifacts (TSO and RT artifacts) as well as differentiating barcodes on multiplexed samples. Iso-Seq 3 achieves the same output performance in high-quality transcript sequences compared to previous versions while reducing the runtime and memory usage dramatically.

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

High-quality human genomes achieved through HiFi sequence data and FALCON-Unzip assembly

De novo assemblies of human genomes from accurate (85-90%), continuous long reads (CLR) now approach the human reference genome in contiguity, but the assembly base pair accuracy is typically below QV40 (99.99%), an order-of-magnitude lower than the standard for finished references. The base pair errors complicate downstream interpretation, particularly false positive indels that lead to false gene loss through frameshifts. PacBio HiFi sequence data, which are both long (>10 kb) and very accurate (>99.9%) at the individual sequence read level, enable a new paradigm in human genome assembly. Haploid human assemblies using HiFi data achieve similar contiguity to those using CLR data and are highly accurate at the base level1. Furthermore, HiFi assemblies resolve more high-identity sequences such as segmental duplications2. To enable HiFi assembly in diploid human samples, we have extended the FALCON-Unzip assembler to work directly with HiFi reads. Here we present phased human diploid genome assemblies from HiFi sequencing of HG002, HG005, and the Vertebrate Genome Project (VGP) mHomSap1 trio on the PacBio Sequel II System. The HiFi assemblies all exceed the VGP’s quality guidelines, approaching QV50 (99.999%) accuracy. For HG002, 60% of the genome was haplotype-resolved, with phase-block N50 of 143Kbp and phasing accuracy of 99.6%. The overall mean base accuracy of the assembly was QV49.7. In conclusion, HiFi data show great promise towards complete, contiguous, and accurate diploid human assemblies.

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 15-20kb reads with average read quality above 99%. Materials and Methods: We sequenced a library from human reference sample HG002 to 18-fold coverage on the PacBio Sequel II with two SMRT Cells 8M. The CCS algorithm was used to generate highly accurate (average 99.9%) 12.9kb 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 99.70% and 96.59% against the GIAB truth set, and pbsv achieves 97.72% recall on structural variants longer than 50bp. Using WhatsHap, small variants were phased into haplotype blocks with 145kb 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

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.