Video Gallery

In this ASHG 2020 PacBio Workshop Emily Farrow of Children’s Mercy Kansas City shares how the incorporation of long-read sequencing into the Genomic Answers for Kids research study is increasing diagnostic yields through the identification of novel genetic variation. Emily highlights several cases in which PacBio HiFi sequencing was able to provide insights where short-read sequencing alone was inconclusive, due to limitations stemming from repetitive regions and large structural variants.

In this ASHG 2020 CoLab presentation hear Principal Scientists, Aaron Wenger and Elizabeth Tseng share how highly accurate long reads (HiFi reads) provide comprehensive variant detection for both genomes and transcriptomes. Aaron Wenger describes how new improvements in protocols and analysis methods have increased scalability and accuracy of variant calling. As demonstrated in the precisionFDA Truth Challenge V2, HiFi reads (>99% accurate, 15 kb – 20 kb) now outperform short reads for single nucleotide and structural variant calling and match for small indels. This includes calling >30,000 small variants and >10,000 structural variants missed by short reads, many in medically relevant genes. Elizabeth Tseng descried how for Single-Cell RNA sequencing, HiFi reads allow for precise detection of UMIs and single cell barcodes while still providing full-length transcript isoform information. Any single cell platform that produces full-length cDNA can be sequenced on the PacBio Systems, with the Sequel II System generating up to 3 million full-length reads per SMRT Cell 8M, sufficient to characterize ~3,000 single cells.

In this ASHG 2020 PacBio Workshop Hagen Tilgner of Cornell University shares how he has used single-cell RNA sequencing using long reads to identify novel isoform expression in brain tissues.

In this ASHG 2020 PacBio Workshop Jonas Korlach, CSO, shares how the new PacBio Sequel IIe System makes highly accurate long-read sequencing easy and affordable so?all scientists can gain comprehensive views of human genomes and transcriptomes. He goes on to provide updates on the applications including human WGS for variant detection, de novo genome assembly, single-cell full-length RNA sequencing, and targeted sequencing using PCR and No-Amp methods.

Introduction: Around 5% (1,168) of protein-coding genes in the human genome contain an exon that is difficult to map with typical next-generation sequencing (NGS) read lengths due to homologous pseudogenes or segmental duplications. Among the difficult-to-map genes are 193 with known medical relevance, including CYP2D6, GBA, SMN1/2, and VWF. Long-read DNA sequencing provides increased mappability, accessing many of the difficult-to-map regions by connecting the homologous exon to neighboring unique sequence. Until recently, the read-level accuracy of long-read sequencing had made it challenging to accurately call small variants. The recently developed HiFi reads from the PacBio Sequel II System provide both long read length (15 kb – 25 kb) for mappability and high read quality (>99%) for accurate variant calling, expanding the regions of the genome that are able to be characterized to high precision and recall. Materials and Methods: Human reference sample HG002 was sequenced to 35-fold HiFi read coverage on the PacBio Sequel II System. Matched 35-fold coverage with NGS reads was obtained on the Illumina NovaSeq 6000. Reads were mapped to the GRCh38 reference genome using pbmm2 for HiFi reads and BWA for NGS reads. Small variants were called using DeepVariant. The variant callsets were compared to each other and to the Genome in a Bottle (GIAB) v4.1 benchmark within exons previously reported to be problematic for NGS. Results: For difficult-to-map exons within the GIAB benchmark, HiFi reads detect 1,269 true benchmark variants, 21% more than are detected with NGS reads (1,053). Small variant precision in difficult-to-map exons is 97.7% for HiFi reads, markedly higher than 92.0% for NGS reads. Extending outside of the benchmark, HiFi reads detect 241 small variants missed by NGS reads across 42 difficult-to-map exons of medically relevant genes, including 14 variants in C4A, 5 in SMN1, and 2 in STRC. Conclusion: HiFi reads have both high mappability and high read quality, which enables accurate small variant calling in difficult-to-map genes that are challenging for NGS. We predict that large-scale use of HiFi reads in disease cohort studies will discover additional disease genes and variants that have remained beyond the reach of NGS.

Although PCR is a cost-effective way to enrich for genomic regions of interest for DNA sequencing, amplifying regions with extreme GC-content and long stretches of short tandem repeat (STR) sequences is often problematic and prone to sequence artifacts. This is especially true when developing multiplexed PCR assays for clinical applications such as carrier screening for multiple genes. The additional challenge is that all PCR primer pairs must be carefully selected to be compatible based on amplicon size and PCR conditions. Due to these experimental design constraints, a single tube with a high number of multiplexed PCR amplicons is difficult to achieve. We have developed an amplification-free enrichment method harnessing the CRISPR-Cas9 system to target hard-to-amplify genomic regions, circumventing potential PCR artifacts for PacBio highly accurate long-read (HiFi) sequencing using this No-Amp Targeted Sequencing approach. The method utilizes a pair of guide RNAs to target and excise each region of interest. This allows simple experimental design for multiplexing targets with variable GC-content and length. In addition, since the method is amplification-free, the DNA base modifications are also preserved. In this study, we show successful enrichment and sequencing of a Thalassemia-a/ß panel for HBA/HBB gene variation detection as well as an Ataxia panel targeting the disease-causing repeat expansion regions in 15 Ataxia genes. In addition to multiplexing several targets, we have also multiplexed at least 20 samples in one experiment making the No-Amp Targeted Sequencing method a cost-effective option. By removing the need for employing multiple assays, our results show the versatility of the method and the potential for using it for various genetic disease carrier screening applications in the future.

Recent advances in sequencing chemistry and software in the Sequel II System enable generating highly accurate long reads that are up to 25 kb in length with >99% accuracy. The high quality HiFi reads are suitable for variant detection of all types, from single nucleotides to structural variants. PacBio offers an end-to-end solution from sample preparation to data analysis. However, library construction is still a bottleneck making it difficult to implement into a high-throughput workflow for sequencing large number of samples. Input DNA requirements, DNA shearing and size-selection/fractionation are the most critical and challenging steps in the current procedure. In this study we will address each of these steps and highlight alternate methods that can be implemented to make the procedure scalable for any automated liquid handling systems. At present, the HiFi library preparation workflow requires 10-15 µg of human genomic DNA, which may be prohibitive for WGS projects where source of DNA is scarce (e.g., DNA extracted from infant blood). As a solution, we have optimized the workflow using 1-2 µg of human genomic DNA generating adequate data with >20-fold genome coverage per sample. DNA was fragmented to 15 kb – 20 kb using Diagenode’s Megaruptor 3 which can process up to 8 samples simultaneously. In addition, library construction using the SMRTbell Express Template Kit 2.0 ensured increased sample recovery with faster turn-around time. Above all, we eliminated the time-consuming size-selection process and employed AMPure PB beads for size-selection making it amenable to automation. The resulting HiFi SMRTbell library generated >24 GB HiFi reads per Sequel II SMRT Cell 8M. The mean CCS read lengths were ~20 kb with read quality of Q31 (median).

COVID-19 is caused by the infection of SARS-CoV-2, a member of the coronavirus family. Complete and accurate sequencing of the SARS-CoV-2 genome enables discovery and epidemiological tracing of mutations that may be important for antiviral and vaccine research. A complementary approach, sequencing the patients’ immune repertoire, allows for detection of neutralizing antibodies and understanding variation in the adaptive immune response. PacBio’s SMRT Sequencing uses circular consensus sequencing that can generate long, highly accurate (HiFi) reads. We find that a tiled multiplex PCR amplicon approach of ~1-2 kb fragments achieves a balanced tradeoff between ease of library preparation and robustness to RNA quality. When mapped against the reference genome, SNPs can be called accurately at as low as 20-fold coverage, meaning that 500+ patient samples can be multiplexed and sequenced on a single SMRT Cell 8M. Higher coverage is required to identify minor variants. Testing different variant callers, we found PacBio’s own MinorVariant (originally developed for HIV quasi-species detection) had the highest sensitivity and specificity. In addition to sequencing the entire 30 kb of the viral genome, we also validated two alternative methods: sequencing only the full-length spike (S) gene in a single 4 kb amplicon, and using probe hybridization to enrich for viral RNAs. The former approach can be utilized to monitor S gene mutations in a high-throughput manner, while the latter approach is robust to sample quality and can readily identify sub-genomic viral RNA fusion events. Sequencing the host immune response in recovered patients is another focal point of COVID-19 research. Here, we show how using a single SMRT Cell 8M with an off-the-shelf Takara SMARTer Human BCR IgG IgM H/K/L Profiling Kit, the PacBio data identifies largely the same clonal types as matching Illumina libraries. However, using an alternative IgG subtype primer that generates a >600 bp amplicon that exceeds the capabilities of PE assembly enables the co-determination of IgG subclass information. Furthermore, the high accuracy of the HiFi reads eliminates the need for IMGT database-assisted assembly; all sequences are generated de novo. Our work demonstrates the feasibility of using PacBio on recovered patients’ peripheral blood lymphocytes RNA for the purpose of identifying neutralizing antibodies.

Most genes in eukaryotic organisms produce alternative isoforms, broadening the diversity of proteins and non-coding RNAs encoded by the genome. In contrast to other RNA sequencing platforms that rely on short-read sequencing, long accurate reads from PacBio Single Molecule, Real-Time (SMRT) Sequencing can characterize full-length transcripts without the need for assembly and inference. The PacBio isoform sequencing (Iso-Seq) method generates full-length sequences for transcripts up to 10 kb in length, with scalable throughput using barcoding approaches. The Iso-Seq application can be employed for a wide variety of studies, including improvement of gene annotation, identification of novel isoforms and fusion transcripts, and differential isoform expression across tissues and cell types. Long-read sequencing together with hybridization-capture targeting provides a powerful approach to target candidate transcripts of interest using biotinylated capture probes. In addition to the comprehensive isoform characterization that offers for targeted genes, long reads provide other advantages as well. For example, our studies revealed that a customized Alzheimer’s Disease (AD) panel captured cDNA applied to two AD subjects could identify heterozygous variants in the targeted genes, allowing phasing of transcript isoforms and sorting into their respective haplotypes using the IsoPhase pipeline. We will also discuss how we rapidly applied the capture long-read RNA-seq concept in recent SARS-CoV2 viral sequencing efforts. Although PCR is widely used in COVID-19 studies, primer design and PCR conditions are susceptible to sample quality and viral titer, resulting in uneven coverage and dropouts. Here we present an alternative approach with probe-based enrichment and long-read sequencing. Viral transcripts in the RNA sample are reverse transcribed into cDNA just as in the standard Iso-Seq workflow but then a custom panel of IDT xGen Lockdown probes tilling the SARS-CoV-2 viral genome are used for capture. The method affords complete genomic sequence determination, shows more even coverage than traditional RT-PCR, and is robust to RNA quality and quantity. Furthermore, we took advantage of unique molecular indexes (UMIs) to separate founder molecules and detect PCR artifacts during sample preparation. This work provides an orthogonal approach for researchers elucidating the virology of this novel coronavirus and we foresee that this workflow can be easily modified to capture long read sequences for other viruses as well.

In this webinar, scientists from PacBio share how using Single Molecule, Real-Time (SMRT) Sequencing, you can generate highly accurate long reads – HiFi reads – with 99% accuracy (Q20) and read lengths of 10 kb or more. This high resolution of each single molecule enables species or strain-level profiling of complex populations in both targeted and shotgun sequencing experiments. Genome assemblies are more cost effective than ever before when sequencing metagenomics samples with the Sequel II System.