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June 1, 2021  |  

SMRT Sequencing solutions for plant genomes and transcriptomes

Single Molecule, Real-Time (SMRT) Sequencing provides efficient, streamlined solutions to address new frontiers in plant genomes and transcriptomes. Inherent challenges presented by highly repetitive, low-complexity regions and duplication events are directly addressed with multi- kilobase read lengths exceeding 8.5 kb on average, with many exceeding 20 kb. Differentiating between transcript isoforms that are difficult to resolve with short-read technologies is also now possible. We present solutions available for both reference genome and transcriptome research that best leverage long reads in several plant projects including algae, Arabidopsis, rice, and spinach using only the PacBio platform. Benefits for these applications are further realized with consistent use of size-selection of input sample using the BluePippin™ device from Sage Science. We will share highlights from our genome projects using the latest P5- C3 chemistry to generate high-quality reference genomes with the highest contiguity, contig N50 exceeding 1 Mb, and average base quality of QV50. Additionally, the value of long, intact reads to provide a no-assembly approach to investigate transcript isoforms using our Iso-Seq protocol will be presented for full transcriptome characterization and targeted surveys of genes with complex structures. PacBio provides the most comprehensive assembly with annotation when combining offerings for both genome and transcriptome research efforts. For more focused investigation, PacBio also offers researchers opportunities to easily investigate and survey genes with complex structures.

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June 1, 2021  |  

Data release for polymorphic genome assembly algorithm development.

Heterozygous and highly polymorphic diploid (2n) and higher polyploidy (n > 2) genomes have proven to be very difficult to assemble. One key to the successful assembly and phasing of polymorphic genomics is the very long read length (9-40 kb) provided by the PacBio RS II system. We recently released software and methods that facilitate the assembly and phasing of genomes with ploidy levels equal to or greater than 2n. In an effort to collaborate and spur on algorithm development for assembly and phasing of heterozygous polymorphic genomes, we have recently released sequencing datasets that can be used to test and develop highly polymorphic diploid and polyploidy assembly and phasing algorithms. These data sets include multiple species and ecotypes of Arabidopsis that can be combined to create synthetic in-silico F1 hybrids with varying levels of heterozygosity. Because the sequence of each individual line was generated independently, the data set provides a ‘ground truth’ answer for the expected results allowing the evaluation of assembly algorithms. The sequencing data, assembly of inbred and in-silico heterozygous samples (n=>2) and phasing statistics will be presented. The raw and processed data has been made available to aid other groups in the development of phasing and assembly algorithms.

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June 1, 2021  |  

Unique haplotype structure determination in human genome using Single Molecule, Real-Time (SMRT) Sequencing of targeted full-length fosmids.

Determination of unique individual haplotypes is an essential first step toward understanding how identical genotypes having different phases lead to different biological interpretations of function, phenotype, and disease. Genome-wide methods for identifying individual genetic variation have been limited in their ability to acquire phased, extended, and complete genomic sequences that are long enough to assemble haplotypes with high confidence. We explore a recombineering approach for isolation and sequencing of a tiling of targeted fosmids to capture interesting regions from human genome. Each individual fosmid contains large genomic fragments (~35?kb) that are sequenced with long-read SMRT technology to generate contiguous long reads. These long reads can be easily de novo assembled for targeted haplotype resolution within an individual’s genomes. The P5-C3 chemistry for SMRT Sequencing generated contiguous, full-length fosmid sequences of 30 to 40 kb in a single read, allowing assembly of resolved haplotypes with minimal data processing. The phase preserved in fosmid clones spanned at least two heterozygous variant loci, providing the essential detail of precise haplotype structures. We show complete assembly of haplotypes for various targeted loci, including the complex haplotypes of the KIR locus (~150 to 200 kb) and conserved extended haplotypes (CEHs) of the MHC region. This method is easily applicable to other regions of the human genome, as well as other genomes.

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June 1, 2021  |  

Evaluation of multiplexing strategies for HLA genotyping using PacBio Sequencing technology.

Fully phased allele-level sequencing of highly polymorphic HLA genes is greatly facilitated by SMRT Sequencing technology. In the present work, we have evaluated multiple DNA barcoding strategies for multiplexing several loci from multiple individuals, using three different tagging methods. Specifically MHC class I genes HLA-A, -B, and –C were indexed via DNA Barcodes by either tailed primers or barcoded SMRTbell adapters. Eight different 16-bp barcode sequences were used in symmetric & asymmetric pairing. Eight DNA barcoded adapters in symmetric pairing were independently ligated to a pool of HLA-A, -B and –C for eight different individuals, one at a time and pooled for sequencing on a single SMRT Cell. Amplicons generated from barcoded primers were pooled upfront for library generation. Eight symmetric barcoded primers were generated for HLA class I genes. These primers facilitated multiplexing of 8 samples and also allowed generation of unique asymmetric pairings for simultaneous amplification from 28 reference genomic DNA samples. The data generated from all 3 methods was analyzed using LAA protocol in SMRT analysis V2.3. Consensus sequences generated were typed using GenDx NGS engine HLA-typing software.

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June 1, 2021  |  

Resolving the ‘dark matter’ in genomes.

Second-generation sequencing has brought about tremendous insights into the genetic underpinnings of biology. However, there are many functionally important and medically relevant regions of genomes that are currently difficult or impossible to sequence, resulting in incomplete and fragmented views of genomes. Two main causes are (i) limitations to read DNA of extreme sequence content (GC-rich or AT-rich regions, low complexity sequence contexts) and (ii) insufficient read lengths which leave various forms of structural variation unresolved and result in mapping ambiguities.

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June 1, 2021  |  

A workflow for the analysis of contigs from the metagenomic shotgun assembly of SMRT Sequencing data

The throughput of SMRT Sequencing and long reads allows microbial communities to be analyzed using a shotgun sequencing approach. Key to leveraging this data is the ability to cluster sequences belonging to the same member of a community. Long reads of up to 40 kb provide a unique capability in identifying those relationships, and pave the way towards finished assemblies of community members. Long reads are highly valuable when samples are more complex and containing lower intra-species variation, such as a larger number of closely related species, or high intra-species variation. Here, we present a collection of tools tailored for the analysis of PacBio metagenomic assemblies. These tools allow for improvements in the assembly results, and greater insight into the complexity of the study communities. Supervised classification is applied to a large set of sequence characteristics (e.g. GC content, raw read coverage, k-mer frequency, and gene prediction information) and to cluster contigs from single or highly related species. Assembly in isolation of the raw data associated with these contigs is shown to improve assembly statistics. A unique feature of SMRT Sequencing is the availability to leverage simultaneously collected base modification / methylation data to aid the clustering of contigs expected to comprise a single or very closely related species. We demonstrate the added value of base modification information to distinguish and study variation within metagenomic samples based on differences in the methylated DNA motifs involved in the restriction modification system. Application of these techniques is demonstrated on a mock community and monkey intestinal microbiome sample.

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June 1, 2021  |  

Resources for advanced bioinformaticians working in plant and animal genomes with SMRT Sequencing.

Significant advances in bioinformatics tool development have been made to more efficiently leverage and deliver high-quality genome assemblies with PacBio long-read data. Current data throughput of SMRT Sequencing delivers average read lengths ranging from 10-15 kb with the longest reads exceeding 40 kb. This has resulted in consistent demonstration of a minimum 10-fold improvement in genome assemblies with contig N50 in the megabase range compared to assemblies generated using only short- read technologies. This poster highlights recent advances and resources available for advanced bioinformaticians and developers interested in the current state-of-the-art large genome solutions available as open-source code from PacBio and third-party solutions, including HGAP, MHAP, and ECTools. Resources and tools available on GitHub are reviewed, as well as datasets representing major model research organisms made publically available for community evaluation or interested developers.

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June 1, 2021  |  

Impact of DNA quality on PacBio RS II read lengths.

Maximizing the read length of next generation sequencing (NGS) facilitates de novo genome assembly. Currently, the PacBio RS II system leads the industry with respect to maximum possible NGS read lengths. Amplicon Express specializes in preparation of high molecular weight, NGS-grade genomic DNA for a variety of applications, including next generation sequencing. This study was performed to evaluate the effects of gDNA quality on PacBio RS II read length.

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June 1, 2021  |  

Complete microbial genomes, epigenomes, and transcriptomes using long-read PacBio Sequencing.

For comprehensive metabolic reconstructions and a resulting understanding of the pathways leading to natural products, it is desirable to obtain complete information about the genetic blueprint of the organisms used. Traditional Sanger and next-generation, short-read sequencing technologies have shortcomings with respect to read lengths and DNA-sequence context bias, leading to fragmented and incomplete genome information. The development of long-read, single molecule, real-time (SMRT) DNA sequencing from Pacific Biosciences, with >10,000 bp average read lengths and a lack of sequence context bias, now allows for the generation of complete genomes in a fully automated workflow. In addition to the genome sequence, DNA methylation is characterized in the process of sequencing. PacBio® sequencing has also been applied to microbial transcriptomes. Long reads enable sequencing of full-length cDNAs allowing for identification of complete gene and operon sequences without the need for transcript assembly. We will highlight several examples where these capabilities have been leveraged in the areas of industrial microbiology, including biocommodities, biofuels, bioremediation, new bacteria with potential commercial applications, antibiotic discovery, and livestock/plant microbiome interactions.

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June 1, 2021  |  

Single Molecule, Real-Time sequencing of full-length cDNA transcripts uncovers novel alternatively spliced isoforms.

In higher eukaryotic organisms, the majority of multi-exon genes are alternatively spliced. Different mRNA isoforms from the same gene can produce proteins that have distinct properties such as structure, function, or subcellular localization. Thus, the importance of understanding the full complement of transcript isoforms with potential phenotypic impact cannot be underscored. While microarrays and other NGS-based methods have become useful for studying transcriptomes, these technologies yield short, fragmented transcripts that remain a challenge for accurate, complete reconstruction of splice variants. The Iso-Seq protocol developed at PacBio offers the only solution for direct sequencing of full-length, single-molecule cDNA sequences to survey transcriptome isoform diversity useful for gene discovery and annotation. Knowledge of the complete isoform repertoire is also key for accurate quantification of isoform abundance. As most transcripts range from 1 – 10 kb, fully intact RNA molecules can be sequenced using SMRT Sequencing (avg. read length: 10-15 kb) without requiring fragmentation or post-sequencing assembly. Our open-source computational pipeline delivers high-quality, non-redundant sequences for unambiguous identification of alternative splicing events, alternative transcriptional start sites, polyA tail, and gene fusion events. The standard Iso-Seq protocol workflow available for all researchers is presented using a deep dataset of full- length cDNA sequences from the MCF-7 cancer cell line, and multiple tissues (brain, heart, and liver). Detected novel transcripts approaching 10 kb and alternative splicing events are highlighted. Even in extensively profiled samples, the method uncovered large numbers of novel alternatively spliced isoforms and previously unannotated genes.

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June 1, 2021  |  

Targeted SMRT Sequencing and phasing using Roche NimbleGen’s SeqCap EZ enrichment

As a cost-effective alternative to whole genome human sequencing, targeted sequencing of specific regions, such as exomes or panels of relevant genes, has become increasingly common. These methods typically include direct PCR amplification of the genomic DNA of interest, or the capture of these targets via probe-based hybridization. Commonly, these approaches are designed to amplify or capture exonic regions and thereby result in amplicons or fragments that are a few hundred base pairs in length, a length that is well-addressed with short-read sequencing technologies. These approaches typically provide very good coverage and can identify SNPs in the targeted region, but are unable to haplotype these variants. Here we describe a targeted sequencing workflow that combines Roche NimbleGen’s SeqCap EZ enrichment technology with Pacific Biosciences’ SMRT Sequencing to provide a more comprehensive view of variants and haplotype information over multi-kilobase regions. While the SeqCap EZ technology is typically used to capture 200 bp fragments, we demonstrate that 6 kb fragments can also be utilized to enrich for long fragments that extend beyond the targeted capture site and well into (and often across) the flanking intronic regions. When combined with the long reads of SMRT Sequencing, multi-kilobase regions of the human genome can be phased and variants detected in exons, introns and intergenic regions.

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June 1, 2021  |  

SMRT Sequencing of DNA and RNA samples extracted from formalin-fixed and paraffin embedded tissues using adaptive focused acoustics by Covaris.

Recent advances in next-generation sequencing have led to an increased use of formalin-fixed and paraffin-embedded (FFPE) tissues for medical samples in disease and scientific research. Single Molecule, Real-Time (SMRT) Sequencing offers a unique advantage for direct analysis of FFPE samples without amplification. However, obtaining ample long-read information from FFPE samples has been a challenge due to the quality and quantity of the extracted DNA. FFPE samples often contain damaged sites, including breaks in the backbone and missing or altered nucleotide bases, which directly impact sequencing and target enrichment. Additionally, the quality and quantity of the recovered DNA vary depending on the extraction methods used. We have evaluated the Covaris® Adaptive Focused Acoustics (AFA) system as a method for obtaining high molecular weight DNA suitable for SMRTbell™ template preparation and subsequent PacBio RS II sequencing. To test the Covaris system, we extracted DNA from normal kidney FFPE scrolls acquired from the Cooperative Human Tissue Network (CHTN), University of Pennsylvania. Damaged sites in the extracted DNA were repaired using a DNA Damage Repair step, and the treated DNA was constructed into SMRTbell libraries for sequencing on the PacBio System. Using the same repaired DNA, we also tested the efficiency of PCR in amplifying targets of up to 10 kb. The resulting amplicons were also constructed into SMRTbell templates for full-length sequencing on the PacBio System. We found the Adaptive Focused Acoustics (AFA) system by Covaris to be effective. This system is easy and simple to use, and the resulting DNA is compatible with SMRTbell library preparation for targeted and whole genome SMRT Sequencing. The data presented here demonstrates feasibility of SMRT Sequencing with FFPE samples.

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June 1, 2021  |  

Full-length HIV-1 env deep sequencing in a donor with broadly neutralizing V1/V2 antibodies.

Background: Understanding the co-evolution of HIV populations and broadly neutralizing antibodies (bNAbs) may inform vaccine design. Novel long-read, next-generation sequencing methods allow, for the first time, full-length deep sequencing of HIV env populations. Methods: We longitudinally examined HIV-1 env populations (12 time points) in a subtype A infected individual from the IAVI primary infection cohort (Protocol C) who developed bNAbs (62% ID50>50 on a diverse panel of 105 viruses) targeting the V1/V2 loop region. We developed a PacBio single molecule, real-time sequencing protocol to deeply sequence full-length env from HIV RNA. Bioinformatics tools were developed to align env sequences, infer phylogenies, and interrogate escape dynamics of key residues and glycosylation sites. PacBio env sequences were compared to env sequences generated through amplification and cloning. Env dynamics and viral escape motif evolution were interpreted in the context of the development V1/V2-targeting broadly neutralizing antibodies. Results: We collected a median of 6799 (range: 1770-14727) high quality full-length HIV env circular consensus sequences (CCS) per SMRT Cell, per time point. Using only CCS reads comprised of 6 or more passes over the HIV env insert (= 16 kb read length) ensured that our median per-base accuracy was 99.7%. A phylogeny inferred with PacBio and 100 cloned env sequences (10 time points) found the cloned sequences evenly distributed among PacBio sequences. Viral escape from the V1/V2 targeted bNAbs was evident at V2 positions 160, 166, 167, 169 and 181 (HxB2 numbering), exhibiting several distinct escape pathways by 40 months post-infection. Conclusions: Our PacBio full-length env sequencing method allowed unprecedented view and ability to characterize HIV-1 env dynamics throughout the first four years of infection. Longitudinal full-length env deep sequencing allows accurate phylogenetic inference, provides a detailed picture of escape dynamics in epitope regions, and can identify minority variants, all of which will prove critical for increasing our understanding of how env evolution drives the development of antibody breadth.

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June 1, 2021  |  

Best practices for whole-genome de novo sequencing with long-read SMRT Sequencing.

With the introduction of P6-C4 chemistry, PacBio has made significant strides with Single Molecule, Real-Time (SMRT) Sequencing . Read lengths averaging between 10 and 15 kb can be now be achieved with extreme reads in the distribution of > 60 kb. The chemistry attains a consensus accuracy of 99.999% (QV50) at 30x coverage which coupled with an increased throughput from the PacBio RS II platform (500 Mb – 1 Gb per SMRT Cell) makes larger genome projects more tractable. These combined advancements in technology deliver results that rival the quality of Sanger “clone-by-clone” sequencing efforts; resulting in closed microbial genomes and highly contiguous de novo assembly of complex eukaryotes on multi-Gbase scale using SMRT Sequencing as the standalone technology. We present here the guidelines and best practices to achieve optimal results when employing PacBio-only whole genome shotgun sequencing strategies. Specific sequencing examples for plant and animal genomes are discussed with SMRTbell library preparation and purification methods for obtaining long insert libraries to generate optimal sequencing results. The benefits of long reads are demonstrated by the highly contiguous assemblies yielding contig N50s of over 5 Mb compared to similar assemblies using next-generation short-read approaches. Finally, guidelines will be presented for planning out projects for the de novo assembly of large genomes.

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June 1, 2021  |  

Full-length cDNA sequencing of alternatively spliced isoforms provides insight into human diseases.

The majority of human genes are alternatively spliced, making it possible for most genes to generate multiple proteins. The process of alternative splicing is highly regulated in a developmental-stage and tissue-specific manner. Perturbations in the regulation of these events can lead to disease in humans. Alternative splicing has been shown to play a role in human cancer, muscular dystrophy, Alzheimer’s, and many other diseases. Understanding these diseases requires knowing the full complement of mRNA isoforms. Microarrays and high-throughput cDNA sequencing have become highly successful tools for studying transcriptomes, however these technologies only provide small fragments of transcripts and building complete transcript isoforms has been very challenging. We have developed the Iso-Seq technique, which is capable of sequencing full-length, single-molecule cDNA sequences. The method employs SMRT Sequencing to generate individual molecules with average read lengths of more than 10 kb and some as long as 40 kb. As most transcripts are from 1 to 10 kb, we can sequence through entire RNA molecules, requiring no fragmentation or post-sequencing assembly. Jointly with the sequencing method, we developed a computational pipeline that polishes these full-length transcript sequences into high-quality, non-redundant transcript consensus sequences. Iso-Seq sequencing enables unambiguous identification of alternative splicing events, alternative transcriptional start and poly-A sites, and transcripts from gene fusion events. Knowledge of the complete set of isoforms from a sample of interest is key for accurate quantification of isoform abundance when using any technology for transcriptome studies. Here we characterize the full-length transcriptome of normal human tissues, paired tumor/normal samples from breast cancer, and a brain sample from a patient with Alzheimer’s using deep Iso-Seq sequencing. We highlight numerous discoveries of novel alternatively spliced isoforms, gene-fusions events, and previously unannotated genes that will improve our understanding of human diseases.

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