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June 1, 2021

An interactive workflow for the analysis of contigs from the metagenomic shotgun assembly of SMRT Sequencing data.

The data throughput of next-generation sequencing allows whole microbial communities to be analyzed using a shotgun sequencing approach. Because a key task in taking advantage of these data is the ability to cluster reads that belong to the same member in a community, single-molecule long reads of up to 30 kb from SMRT Sequencing provide a unique capability in identifying those relationships and pave the way towards finished assemblies of community members. Long reads become even more valuable as samples get more complex with lower intra-species variation, a larger number of closely related species, or high intra-species variation. Here we present a collection of tools tailored for PacBio data for the analysis of these fragmented metagenomic assembles, allowing improvements in the assembly results, and greater insight into the communities themselves. 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, allowing the clustering of contigs from single or highly related species. A unique feature of SMRT Sequencing data is the availability of base modification / methylation information, which can be used to further analyze clustered contigs expected to be comprised of single or very closely related species. Here we show base modification information can be used to further study variation, based on differences in the methylated DNA motifs involved in the restriction modification system. Application of these techniques is demonstrated on a monkey intestinal microbiome sample and an in silico mix of real sequencing data from distinct bacterial samples.

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June 1, 2021

Developments in PacBio metagenome sequencing: Shotgun whole genomes and full-length 16S.

The assembly of metagenomes is dramatically improved by the long read lengths of SMRT Sequencing. This is demonstrated in an experimental design to sequence a mock community from the Human Microbiome Project, and assemble the data using the hierarchical genome assembly process (HGAP) at Pacific Biosciences. Results of this analysis are promising, and display much improved contiguity in the assembly of the mock community as compared to publicly available short-read data sets and assemblies. Additionally, the use of base modification information to make further associations between contigs provides additional data to improve assemblies, and to distinguish between members within a microbial community. The epigenetic approach is a novel validation method unique to SMRT Sequencing. In addition to whole-genome shotgun sequencing, SMRT Sequencing also offers improved classification resolution and reliability of metagenomic and microbiome samples by the full-length sequencing of 16S rRNA (~1500 bases long). Microbial communities can be detected at the species level in some cases, rather than being limited to the genus taxonomic classification as constrained by short-read technologies. The performance of SMRT Sequencing for these metagenomic samples achieved >99% predicted concordance to reference sequences in cecum, soil, water, and mock control investigations for bacterial 16S. Community samples are estimated to contain from 2.3 and up to 15 times as many species with abundance levels as low as 0.05% compared to the identification of phyla groups.

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June 1, 2021

SMRT Sequencing solutions for investigative studies to understand evolutionary processes.

Single Molecule, Real-Time (SMRT) Sequencing holds promise for addressing new frontiers to understand molecular mechanisms in evolution and gain insight into adaptive strategies. With read lengths exceeding 10 kb, we are able to sequence high-quality, closed microbial genomes with associated plasmids, and investigate large genome complexities, such as long, highly repetitive, low-complexity regions and multiple tandem-duplication events. Improved genome quality, observed at 99.9999% (QV60) consensus accuracy, and significant reduction of gap regions in reference genomes (up to and beyond 50%) allow researchers to better understand coding sequences with high confidence, investigate potential regulatory mechanisms in noncoding regions, and make inferences about evolutionary strategies that are otherwise missed by the coverage biases associated with short- read sequencing technologies. Additional benefits afforded by SMRT Sequencing include the simultaneous capability to detect epigenomic modifications and obtain full-length cDNA transcripts that obsolete the need for assembly. With direct sequencing of DNA in real-time, this has resulted in the identification of numerous base modifications and motifs, which genome-wide profiles have linked to specific methyltransferase activities. Our new offering, the Iso-Seq Application, allows for the accurate differentiation between transcript isoforms that are difficult to resolve with short-read technologies. PacBio reads easily span transcripts such that both 5’/3’ primers for cDNA library generation and the poly-A tail are observed. As such, exon configuration and intron retention events can be analyzed without ambiguity. This technological advance is useful for characterizing transcript diversity and improving gene structure annotations in reference genomes. We review solutions available with SMRT Sequencing, from targeted sequencing efforts to obtaining reference genomes (>100 Mb). This includes strategies for identifying microsatellites and conducting phylogenetic comparisons with targeted gene families. We highlight how to best leverage our long reads that have exceeded 20 kb in length for research investigations, as well as currently available bioinformatics strategies for analysis. Benefits for these applications are further realized with consistent use of size selection of input sample using the BluePippin™ device from Sage Science as demonstrated in our genome improvement projects. Using the latest P5-C3 chemistry on model organisms, these efforts have yielded an observed contig N50 of ~6 Mb, with the longest contig exceeding 12.5 Mb and an average base quality of QV50.

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June 1, 2021

Genomic DNA sequences of HLA class I alleles generated using multiplexed barcodes and SMRT DNA Sequencing technology.

Allelic-level resolution HLA typing is known to improve survival prognoses post Unrelated Donor (UD) Haematopoietic Stem Cell Transplantation (HSCT). Currently, many commonly used HLA typing methodologies are limited either due to the fact that ambiguity cannot be resolved or that they are not amenable to high-throughput laboratories. Pacific Biosciences’ Single Molecule Real-Time (SMRT) DNA sequencing technology enables sequencing of single molecules in isolation and has read-length capabilities to enable whole gene sequencing for HLA. DNA barcode technology labels samples with unique identifiers that can be traced throughout the sequencing process. The use of DNA barcodes means that multiple samples can be sequenced in a single experiment but data can still be attributed to the correct sample. Here we describe the results of experiments that use DNA barcodes to facilitate sequencing of multiple samples for full-length HLA class I genes (known as multiplexing).

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June 1, 2021

HLA sequencing using SMRT Technology – High resolution and high throughput HLA genotyping in a clinical setting

Sequence based typing (SBT) is considered the gold standard method for HLA typing. Current SBT methods are rather laborious and are prone to phase ambiguity problems and genotyping uncertainties. As a result, the NGS community is rapidly seeking to remedy these challenges, to produce high resolution and high throughput HLA sequencing conducive to a clinical setting. Today, second generation NGS technologies are limited in their ability to yield full length HLA sequences required for adequate phasing and identification of novel alleles. Here we present the use of single molecule real time (SMRT) sequencing as a means of determining full length/long HLA sequences. Moreover we reveal the scalability of this method through multiplexing approches and determine HLA genotyping calls through the use of third party Gendx NGSengine® software.

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June 1, 2021

Long Amplicon Analysis: Highly accurate, full-length, phased, allele-resolved gene sequences from multiplexed SMRT Sequencing data.

The correct phasing of genetic variations is a key challenge for many applications of DNA sequencing. Allele-level resolution is strongly preferred for histocompatibility sequencing where recombined genes can exhibit different compatibilities than their parents. In other contexts, gene complementation can provide protection if deleterious mutations are found on only one allele of a gene. These problems are especially pronounced in immunological domains given the high levels of genetic diversity and recombination seen in regions like the Major Histocompatibility Complex. A new tool for analyzing Single Molecule, Real-Time (SMRT) Sequencing data – Long Amplicon Analysis (LAA) – can generate highly accurate, phased and full-length consensus sequences for multiple genes in a single sequencing run.

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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

Highly sensitive, non-invasive detection of colorectal cancer mutations using single molecule, third generation sequencing.

Colorectal cancer (CRC) represents one of the most prevalent and lethal malignant neoplasms and every individual of age 50 and above should undergo regular CRC screening. Currently, the most effective procedure to detect adenomas, the precursors to CRC, is colonoscopy, which reduces CRC incidence by 80%. However, it is an invasive approach that is unpleasant for the patient, expensive, and poses some risk of complications such as colon perforation. A non-invasive screening approach with detection rates comparable to those of colonoscopy has not yet been established. The current study applies Pacific Biosciences third generation, single molecule sequencing to the inspection of CRC-driving mutations. Our approach combines the screening power and the extremely high accuracy of circular consensus (CCS) third generation sequencing with the non-invasiveness of using stool DNA to detect CRC-associated mutations present at extremely low frequencies and establishes a foundation for a non-invasive, highly sensitive assay to screen the population for CRC and early stage adenomas. We performed a series of experiments using a pool of fifteen amplicons covering the genes most frequently mutated in CRC (APC, Beta Catenin, KRAS, BRAF, and TP53), ensuring a theoretical screening coverage of over 97% for both CRC and adenomas. The assay was able to detect mutations in DNA isolated from stool samples from patients diagnosed with CRC at frequencies below 0.5 % with no false positives. The mutations were then confirmed by sequencing DNA isolated from the excised tumor samples. Our assay should be sensitive enough to allow the early identification of adenomatous polyps using stool DNA as analyte. In conclusion, we have developed an assay to detect mutations in the genes associated with CRC and adenomas using Pacific Biosciences RS Single Molecule, Real Time Circular Consensus Sequencing (SMRT-CCS). With no systematic bias and a much higher raw base-calling quality (CCS) compared to other sequencing methods, the assay was able to detect mutations in stool DNA at frequencies below 0.5 % with no false positives. This level of sensitivity should be sufficient to allow the detection of most adenomatous polyps using stool DNA as analyte, a feature that would make our approach the first non-invasive assay with a sensitivity comparable to that of colonoscopy and a strong candidate for the non-invasive preventive CRC screening of the general population.

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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

Complex alternative splicing patterns in hematopoietic cell subpopulations revealed by third-generation long reads.

Background: Alternative splicing expands the repertoire of gene functions and is a signature for different cell populations. Here we characterize the transcriptome of human bone marrow subpopulations including progenitor cells to understand their contribution to homeostasis and pathological conditions such as atherosclerosis and tumor metastasis. To obtain full-length transcript structures, we utilized long reads in addition to RNA-seq for estimating isoform diversity and abundance. Method: Freshly harvested, viable human bone marrow tissues were extracted from discarded harvesting equipment and separated into total bone marrow (total), lineage-negative (lin-) progenitor cells and differentiated cells (lin+) by magnetic bead sorting with antibodies to surface markers of hematopoietic cell lineages. Sequencing was done with SOLiD, Illumina HiSeq (100bp paired-end reads), and PacBio RS II (full-length cDNA library protocol for 1 – 6 kb libraries). Short reads were assembled using both Trinity for de novo assembly and Cufflinks for genome-guided assembly. Full-length transcript consensus sequences were obtained for the PacBio data using the RS_IsoSeq protocol from PacBios SMRTAnalysis software. Quantitation for each sample was done independently for each sequencing platform using Sailfish to obtain the TPM (transcripts per million) using k-mer matching. Results: PacBios long read sequencing technology is capable of sequencing full-length transcripts up to 10 kb and reveals heretofore-unseen isoform diversity and complexity within the hematopoietic cell populations. A comparison of sequencing depth and de novo transcript assembly with short read, second-generation sequencing reveals that, while short reads provide precision in determining portions of isoform structure and supporting larger 5 and 3 UTR regions, it fails in providing a complete structure especially when multiple isoforms are present at the same locus. Increased breadth of isoform complexity is revealed by long reads that permits further elaboration of full isoform diversity and specific isoform abundance within each separate cell population. Sorting the distribution of major and minor isoforms reveals a cell population-specific balance focused on distinct genome loci and shows how tissue specificity and diversity are modulated by alternative splicing.

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