June 1, 2021  |  

Isoform sequencing: Unveiling the complex landscape of the eukaryotic transcriptome on the PacBio RS II.

Alternative splicing of RNA is an important mechanism that increases protein diversity and is pervasive in the most complex biological functions. While advances in RNA sequencing methods have accelerated our understanding of the transcriptome, isoform discovery remains computationally challenging due to short read lengths. Here, we describe the Isoform Sequencing (Iso-Seq) method using long reads generated by the PacBio RS II. We sequenced rat heart and lung RNA using the Clontech® SMARTer® cDNA preparation kit followed by size selection using agarose gel. Additionally, we tested the BluePippin™ device from Sage Science for efficiently extracting longer transcripts = 3 kb. Post-sequencing, we developed a novel isoform-level clustering algorithm to generate high-quality transcript consensus sequences. We show that our method recovered alternative splice forms as well as alternative stop sites, antisense transcription, and retained introns. To conclude, the Iso-Seq method provides a new opportunity for researchers to study the complex eukaryotic transcriptome even in the absence of reference genomes or annotated transcripts.


June 1, 2021  |  

Accurately surveying uncultured microbial species with SMRT Sequencing

Background: Microbial ecology is reshaping our understanding of the natural world by revealing the large phylogenetic and functional diversity of microbial life. However the vast majority of these microorganisms remain poorly understood, as most cultivated representatives belong to just four phylogenetic groups and more than half of all identified phyla remain uncultivated. Characterization of this microbial ‘dark matter’ will thus greatly benefit from new metagenomic methods for in situ analysis. For example, sensitive high throughput methods for the characterization of community composition and structure from the sequencing of conserved marker genes. Methods: Here we utilize Single Molecule Real-Time (SMRT) sequencing of full-length 16S rRNA amplicons to phylogenetically profile microbial communities to below the genus-level. We test this method on a mock community of known composition, as well as a previously studied microbial community from a lake known to predominantly contain poorly characterized phyla. These results are compared to traditional 16S tag sequencing from short-read technologies and subsets of the full-length data corresponding to the same regions of the 16S gene. Results: We explore the benefits of using full-length amplicons for estimating community structure and diversity. In addition, we investigate the possible effects of context-specific and GC-content biases known to affect short-read sequencing technologies on the predicted community structure. We characterize the potential benefits of profiling metagenomic communities with full-length 16S rRNA genes from SMRT sequencing relative to standard methods.


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.


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.


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.


June 1, 2021  |  

Old school/new school genome sequencing: One step backward — a quantum leap forward.

As the costs for genome sequencing have decreased the number of “genome” sequences have increased at a rapid pace. Unfortunately, the quality and completeness of these so–called “genome” sequences have suffered enormously. We prefer to call such genome assemblies as “gene assembly space” (GAS). We believe it is important to distinguish GAS assemblies from reference genome assemblies (RGAs) as all subsequent research that depends on accurate genome assemblies can be highly compromised if the only assembly available is a GAS assembly.


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.


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.


June 1, 2021  |  

Toward comprehensive genomics analysis with de novo assembly.

Whole genome sequencing can provide comprehensive information important for determining the biochemical and genetic nature of all elements inside a genome. The high-quality genome references produced from past genome projects and advances in short-read sequencing technologies have enabled quick and cheap analysis for simple variants. However even with the focus on genome-wide resequencing for SNPs, the heritability of more than 50% of human diseases remains elusive. For non-human organisms, high-contiguity references are deficient, limiting the analysis of genomic features. The long and unbiased reads from single molecule, real-time (SMRT) Sequencing and new de novo assembly approaches have demonstrated the ability to detect more complicated variants and chromosome-level phasing. Moreover, with the recent advance of bioinformatics algorithms and tools, the computation tasks for completing high-quality de novo assembly of large genomes becomes feasible with commodity hardware. Ongoing development in sequencing technologies and bioinformatics will likely lead to routine generation of high-quality reference assemblies in the future. We discuss the current state of art and the challenges in bioinformatics toward such a goal. More specifically, explicit examples of pragmatic computational requirements for assembling mammalian-size genomes and algorithms suitable for processing diploid genomes are discussed.


June 1, 2021  |  

Building a platinum human genome assembly from single haplotype human genomes generated from long molecule sequencing

The human reference sequence has provided a foundation for studies of genome structure, human variation, evolutionary biology, and disease. At the time the reference was originally completed there were some loci recalcitrant to closure; however, the degree to which structural variation and diversity affected our ability to produce a representative genome sequence at these loci was still unknown. Many of these regions in the genome are associated with large, repetitive sequences and exhibit complex allelic diversity such producing a single, haploid representation is not possible. To overcome this challenge, we have sequenced DNA from two hydatidiform moles (CHM1 and CHM13), which are essentially haploid. CHM13 was sequenced with the latest PacBio technology (P6-C5) to 52X genome coverage and assembled using Daligner and Falcon v0.2 (GCA_000983455.1, CHM13_1.1). Compared to the first mole (CHM1) PacBio assembly (GCA_001007805.1, 54X) contig N50 of 4.5Mb, the contig N50 of CHM13_1.1 is almost 13Mb, and there is a 13-fold reduction in the number of contigs. This demonstrates the improved contiguity of sequence generated with the new chemistry. We annotated 50,188 RefSeq transcripts of which only 0.63% were split transcripts, and the repetitive and segmental duplication content was within the expected range. These data all indicate an extremely high quality assembly. Additionally, we sequenced CHM13 DNA using Illumina SBS technology to 60X coverage, aligned these reads to the GRCh37, GRCh38, and CHM13_1.1 assemblies and performed variant calling using the SpeedSeq pipeline. The number of single nucleotide variants (SNV) and indels was comparable between GRCh37 and GRCh38. Regions that showed increased SNV density in GRCh38 compared to GRCh37 could be attributed to the addition of centromeric alpha satellite sequence to the reference assembly. Alternatively, regions of decreased SNV density in GRCh38 were concentrated in regions that were improved from BAC based sequencing of CHM1 such as 1p12 and 1q21 containing the SRGAP2 gene family. The alignment of PacBio reads to GRCh37 and GRCh38 assemblies allowed us to resolve complex loci such as the MHC region where the best alignment was to the DBB (A2-B57-DR7) haplotype. Finally, we will discuss how combining the two high quality mole assemblies can be used for benchmarking and novel bioinformatics tool development.


June 1, 2021  |  

Improving the goat long-read assembly with optical mapping and Hi-C scaffolding

Reference genome assemblies provide important context in genetics by standardizing the order of genes and providing a universal set of coordinates for individual nucleotides. Often due to the high complexity of genic regions and higher copy number of genes involved in immune function, immunity-related genes are often misassembled in current reference assemblies. This problem is particularly ubiquitous in the reference genomes of non-model organisms as they often do not receive the years of curation necessary to resolve annotation and assembly errors. In this study, we reassemble a reference genome of the goat (Capra hircus) using modern PacBio technology in tandem with BioNano Genomics Irys optical maps and Lachesis clustering in order to provide a high quality reference assembly without the need for extensive filtering. Initial PacBio assemblies using P5C4 chemistry achieved contig N50’s of 4 Megabases and a BUSCO completion score of 84.0%, which is comparable to several finished model organism reference assemblies. We used BioNano Genomics’ Irys platform to generate 336 scaffolds from this data with a scaffold N50 of 24 megabases and total genome coverage of 98%. Lachesis interaction maps were used with a clustering algorithm to associate Irys scaffolds into the expected 30 chromosome physical maps. Comparisons of the initial hybrid scaffolds generated from the long read contigs and optical map information to a previously generated RH map revealed that the entirety of the Goat autosome 20 physical map was contained within one scaffold. Additionally, the BioNano scaffolding resolved several difficult regions that contained genes related to innate immunity which were problem regions in previous reference genome assemblies.


June 1, 2021  |  

Multiplex target enrichment using barcoded multi-kilobase fragments and probe-based capture technologies

Target enrichment capture methods allow scientists to rapidly interrogate important genomic regions of interest for variant discovery, including SNPs, gene isoforms, and structural variation. Custom targeted sequencing panels are important for characterizing heterogeneous, complex diseases and uncovering the genetic basis of inherited traits with more uniform coverage when compared to PCR-based strategies. With the increasing availability of high-quality reference genomes, customized gene panels are readily designed with high specificity to capture genomic regions of interest, thus enabling scientists to expand their research scope from a single individual to larger cohort studies or population-wide investigations. Coupled with PacBio® long-read sequencing, these technologies can capture 5 kb fragments of genomic DNA (gDNA), which are useful for interrogating intronic, exonic, and regulatory regions, characterizing complex structural variations, distinguishing between gene duplications and pseudogenes, and interpreting variant haplotyes. In addition, SMRT® Sequencing offers the lowest GC-bias and can sequence through repetitive regions. We demonstrate the additional insights possible by using in-depth long read capture sequencing for key immunology, drug metabolizing, and disease causing genes such as HLA, filaggrin, and cancer associated genes.


June 1, 2021  |  

Phased human genome assemblies with Single Molecule, Real-Time Sequencing

In recent years, human genomic research has focused on comparing short-read data sets to a single human reference genome. However, it is becoming increasingly clear that significant structural variations present in individual human genomes are missed or ignored by this approach. Additionally, remapping short-read data limits the phasing of variation among individual chromosomes. This reduces the newly sequenced genome to a table of single nucleotide polymorphisms (SNPs) with little to no information as to the co-linearity (phasing) of these variants, resulting in a “mosaic” reference representing neither of the parental chromosomes. The variation between the homologous chromosomes is lost in this representation, including allelic variations, structural variations, or even genes present in only one chromosome, leading to lost information regarding allelic-specific gene expression and function. To address these limitations, we have made significant progress integrating haplotype information directly into genome assembly process with long reads. The FALCON-Unzip algorithm leverages a string graph assembly approach to facilitate identification and separation of heterozygosity during the assembly process to produce a highly contiguous assembly with phased haplotypes representing the genome in its diploid state. The outputs of the assembler are pairs of sequences (haplotigs) containing the allelic differences, including SNPs and structural variations, present in the two sets of chromosomes. The development and testing of our de-novo diploid assembler was facilitated and carefully validated using inbred reference model organisms and F1 progeny, which allowed us to ascertain the accuracy and concordance of haplotigs relative to the two inbred parental assemblies. Examination of the results confirmed that our haplotype-resolved assemblies are “Gold Level” reference genomes having a quality similar to that of Sanger-sequencing, BAC-based assembly approaches. We further sequenced and assembled two well-characterized human samples into their respective phased diploid genomes with gap-free contig N50 sizes greater than 23 Mb and haplotig N50 sizes greater than 380 kb. Results of these assemblies and a comparison between the haplotype sets are presented.


June 1, 2021  |  

Target enrichment using a neurology panel for 12 barcoded genomic DNA samples on the PacBio SMRT Sequencing platform

Target enrichment is a powerful tool for studies involved in understanding polymorphic SNPs with phasing, tandem repeats, and structural variations. With increasing availability of reference genomes, researchers can easily design a cost-effective targeted investigation with custom probes specific to regions of interest. Using PacBio long-read technology in conjunction with probe capture, we were able to sequence multi-kilobase enriched regions to fully investigate intronic and exonic regions, distinguish haplotypes, and characterize structural variations. Furthermore, we demonstrate this approach is advantageous for studying complex genomic regions previously inaccessible through other sequencing platforms. In the present work, 12 barcoded genomic DNA (gDNA) samples were sheared to 6 kb for target enrichment analysis using the Neurology panel provided by Roche NimbleGen. Probe-captured DNA was used to make SMRTbell libraries for SMRT Sequencing on the PacBio RS II. Our results demonstrate the ability to multiplex 12 samples and achieve 1300x enrichment of targeted regions. In addition, we achieved an even representation of on-target rate of 70% across the 12 barcoded genomic DNA samples.


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