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June 1, 2021  |  

No-amp targeted SMRT sequencing using a CRISPR-Cas9 enrichment method

Targeted sequencing of genomic DNA requires an enrichment method to generate detectable amounts of sequencing products. Genomic regions with extreme composition bias and repetitive sequences can pose a significant enrichment challenge. Many genetic diseases caused by repeat element expansions are representative of these challenging enrichment targets. PCR amplification, used either alone or in combination with a hybridization capture method, is a common approach for target enrichment. While PCR amplification can be used successfully with genomic regions of moderate to high complexity, it is the low-complexity regions and regions containing repetitive elements sometimes of indeterminate lengths due to repeat expansions that can lead to poor or failed PCR enrichment. We have developed an enrichment method for targeted SMRT Sequencing on the PacBio Sequel System using the CRISPR-Cas9 system that requires no PCR amplification. Briefly, a preformed SMRTbell library containing the target region of interest is cleaved with Cas9 through direct interaction with a sequence-specific guide RNA. After ligation with new poly(A) hairpin adapters, the asymmetric SMRTbell templates are enriched by magnetic bead separation. This method, paired with SMRT Sequencing’s long reads, high consensus accuracy, and uniform coverage, allows sequencing of genomic regions regardless of challenging sequence context that cannot be investigated with other technologies. The method is amenable to analyzing multiple samples and/or targets in a single reaction. In addition, this method also preserves epigenetic modifications allowing for the detection and characterization of DNA methylation which has been shown to be a key factor in the disease mechanism for some repeat expansion diseases. Here we present results of our latest No-Amp Targeted Sequencing procedure applied to the characterization of CAG triplet repeat expansions in the HTT gene responsible for Huntington’s Disease.

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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.

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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.

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June 1, 2021  |  

Structural variant detection with long read sequencing reveals driver and passenger mutations in a melanoma cell line

Past large scale cancer genome sequencing efforts, including The Cancer Genome Atlas and the International Cancer Genome Consortium, have utilized short-read sequencing, which is well-suited for detecting single nucleotide variants (SNVs) but far less reliable for detecting variants larger than 20 base pairs, including insertions, deletions, duplications, inversions and translocations. Recent same-sample comparisons of short- and long-read human reference genome data have revealed that short-read resequencing typically uncovers only ~4,000 structural variants (SVs, =50 bp) per genome and is biased towards deletions, whereas sequencing with PacBio long-reads consistently finds ~20,000 SVs, evenly balanced between insertions and deletions. This discovery has important implications for cancer research, as it is clear that SVs are both common and biologically important in many cancer subtypes, including colorectal, breast and ovarian cancer. Without confident and comprehensive detection of structural variants, it is unlikely we have a sufficiently complete picture of all the genomic changes that impact cancer development, disease progression, treatment response, drug resistance, and relapse. To begin to address this unmet need, we have sequenced the COLO829 tumor and matched normal lymphoblastoid cell lines to 49- and 51-fold coverage, respectively, with PacBio SMRT Sequencing, with the goal of developing a high-confidence structural variant call set that can be used to empirically evaluate cost-effective experimental designs for larger scale studies and develop structural variation calling software suitable for cancer genomics. Structural variant calling revealed over 21,000 deletions and 19,500 insertions larger than 20 bp, nearly four times the number of events detected with short-read sequencing. The vast majority of events are shared between the tumor and normal, with about 100 putative somatic deletions and 400 insertions, primarily in microsatellites. A further 40 rearrangements were detected, nearly exclusively in the tumor. One rearrangement is shared between the tumor and normal, t(5;X) which disrupts the mismatch repeat gene MSH3, and is likely a driver mutation. Generating high-confidence call sets that cover the entire size-spectrum of somatic variants from a range of cancer model systems is the first step in determining what will be the best approach for addressing an ongoing blind spot in our current understanding of cancer genomes. Here the application of PacBio sequencing to a melanoma cancer cell line revealed thousands of previously overlooked variants, including a mutation likely involved in tumorogenesis.

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June 1, 2021  |  

Comparison of sequencing approaches applied to complex soil metagenomes to resolve proteins of interest

Background: Long-read sequencing presents several potential advantages for providing more complete gene profiling of metagenomic samples. Long reads can capture multiple genes in a single read, and longer reads typically result in assemblies with better contiguity, especially for higher abundance organisms. However, a major challenge with using long reads has been the higher cost per base, which may lead to insufficient coverage of low-abundance species. Additionally, lower single-pass accuracy can make gene discovery for low-abundance organisms difficult. Methods: To evaluate the pros and cons of long reads for metagenomics, we directly compared PacBio and Illumina sequencing on a soil-derived sample, which included spike-in controls of known concentrations of pure referenced samples. For PacBio sequencing, a 10 kb library was sequenced on the Sequel System with 3.0 chemistry. Highly accurate long reads (HiFi reads) with Q20 and higher were generated for downstream analyses using PacBio Circular Consensus Sequencing (CCS) mode. Results were assessed according to the following criteria: DNA extraction capacity, bioinformatics pipeline status, % of proteins with ambiguous AA’s, total unique error-free genes/$1000, total proteins observed in spike-ins/$1000, proteins of interest/$1000, median length of contigs with proteins, and assembly requirements. Results: Both methods had areas of superior performance. DNA extraction capacity was higher for Illumina, the bioinformatics pipeline is well-tested, and there was a lower proportion of proteins with ambiguous AA’s. On the other hand, with PacBio, twice as many unique error-free genes, twice as many total proteins from spike-ins, and ~6 times more proteins of interest were found per $1000 cost. PacBio data produced on average 5 times longer contigs capturing proteins of interest. Additionally, assembly was not required for gene or protein finding, as was the case with Illumina data. Conclusions: In this comparison of PacBio Sequel System with Illumina NextSeq on a complex microbiome, we conclude that the sequencing system of choice may vary, depending on the goals and resources for the project. PacBio sequencing requires a longer DNA extraction method, and the bioinformatics pipeline may require development. On the other hand, the Sequel System generates hundreds of thousands of long HiFi reads per SMRT Cell, producing more genes, more proteins, and longer contigs, thereby offering more information about the metagenomic samples for a lower cost.

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