June 1, 2021  |  

New discoveries from closing Salmonella genomes using Pacific Biosciences continuous long reads.

The newer hierarchical genome assembly process (HGAP) performs de novo assembly using data from a single PacBio long insert library. To assess the benefits of this method, DNA from several Salmonella enterica serovars was isolated from a pure culture. Genome sequencing was performed using Pacific Biosciences RS sequencing technology. The HGAP process enabled us to close sixteen Salmonella subsp. enterica genomes and their associated mobile elements: The ten serotypes include: Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) S. Bareilly, S. Heidelberg, S. Cubana, S. Javiana and S. Typhimurium, S. Newport, S. Montevideo, S. Agona, and S. Tennessee. In addition, we were able to detect novel methyltransferases (MTases) by using the Pacific Biosciences kinetic score distributions showing that each serovar appears to have a novel methylation pattern. For example while all Salmonella serovars examined so far have methylase specific activity for 5’-GATC-3’/3’-CTAG-5’ and 5’-CAGAG-3’/3’-GTCTC-5’ (underlined base indicates a modification), S. Heidelberg is uniquely specific for 5’-ACCANCC-3’/3’-TGGTNGG-5’, while S. Typhimurium has uniquely methylase specific for 5′-GATCAG-3’/3′- CTAGTC-5′ sites, for the samples examined so far. We believe that this may be due to the unique environments and phages that these serotypes have been exposed to. Furthermore, our analysis identified and closed a variety of plasmids such as mobilization plasmids, antimicrobial resistance plasmids and IncX plasmids carrying a Type IV secretion system (T4SS). The VirB/D4 T4SS apparatus is important in that it assists with rapid dissemination of antibiotic resistance and virulence determinants. Presently, only limited information exists regarding the genotypic characterization of drug resistance in S. Heidelberg isolates derived from various host species. Here, we characterize two S. Heidelberg outbreak isolates from two different outbreaks. Both isolates contain the IncX plasmid of approximately 35 kb, and carried the genes virB1, virB2, virB3/4, virB5, virB6, virB7, virB8, virB9, virB10, virB11, virD2, and virD4, that are associated with the T4SS. In addition, the outbreak isolate associated with ground turkey carries a 4,473 bp mobilization plasmid and an incompatibility group (Inc) I1 antimicrobial resistance plasmid encoding resistance to gentamicin (aacC2), beta-lactam (bl2b_tem), streptomycin (aadAI) and tetracycline (tetA, tetR) while the outbreak isolate associated with chicken breast carries the IncI1 plasmid encoding resistance to gentamicin (aacC2), streptomycin (aadAI) and sulfisoxazole (sul1). Using this new technology we explored the genetic elements present in resistant pathogens which will achieve a better understanding of the evolution of Salmonella.


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  |  

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.


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  |  

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.


June 1, 2021  |  

Access full spectrum of polymorphisms in HLA class I & II genes, without imputation for disease association and evolutionary research.

MHC class I and II genes are critically monitored by high-resolution sequencing for organ transplant decisions due to their role in GVHD. Their direct or linkage-based causal association, have increased their prominence as targets for drug sensitivity, autoimmune, cancer and infectious disease research. Monitoring HLA genes can however be tricky due to their highly polymorphic nature. Allele-level resolution is thus strongly preferred. However, most studies were historically focused on peptide binding domains of the HLA genes, due to technological challenges. As a result knowledge about the functional role of polymorphisms outside of exons 2 and 3 of HLA genes was rather limited. There are also relatively few full-length gene references currently available in the IMGT HLA database. This made it difficult to quickly adopt high-throughput reference-reliant methods for allele-level HLA sequencing. Increasing awareness regarding role of regulatory region polymorphisms of HLA genes in disease association1, nonetheless have brought about a revolution in full-length HLA gene sequencing. Researchers are now exploring ways to obtain complete information for HLA genes and integrate it with the current HLA database so it can be interpreted used by clinical researchers. We have explored advantages of SMRT Sequencing to obtain fully phased, allele-specific sequences of HLA class I and II genes for 96 samples using completely De novo consensus generation approach for imputation-free 4-field typing. With long read lengths (average >10 kb) and consensus accuracy exceeding 99.999% (Q50), a comprehensive snapshot of variants in exons, introns and UTRs could be obtained for spectrum of polymorphisms in phase across SNP-poor regions. Such information can provide invaluable insights in future causality association and population diversity research.


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  |  

Genome and transcriptome of the refeneration-competent flatworm, Macrostomum lignano

The free-living flatworm, Macrostomum lignano, much like its better known planarian relative, Schmidtea mediterranea, has an impressive regenerative capacity. Following injury, this species has the ability to regenerate almost an entirely new organism. This is attributable to the presence of an abundant somatic stem cell population, the neoblasts. These cells are also essential for the ongoing maintenance of most tissues, as their loss leads to irreversible degeneration of the animal. This set of unique properties makes a subset of flatworms attractive organisms for studying the evolution of pathways involved in tissue self-renewal, cell fate specification, and regeneration. The use of these organisms as models, however, is hampered by the lack of a well-assembled and annotated genome sequences, fundamental to modern genetic and molecular studies. Here we report the genomic sequence of Macrostomum lignano and an accompanying characterization of its transcriptome. The genome structure of M. lignano is remarkably complex, with ~75% of its sequence being comprised of simple repeats and transposon sequences. This has made high quality assembly from Illumina reads alone impossible (N50=222 bp). We therefore generated 130X coverage by long sequencing reads from the PacBio platform to create a substantially improved assembly with an N50 of 64 Kbp. We complemented the reference genome with an assembled and annotated transcriptome, and used both of these datasets in combination to probe gene expression patterns during regeneration, examining pathways important to stem cell function. As a whole, our data will provide a crucial resource for the community for the study not only of invertebrate evolution and phylogeny but also of regeneration and somatic pluripotency.


June 1, 2021  |  

Comparative Studies of Mammalian Sex Chromosomes: From Cytogenetics to NGS

It is a common knowledge that sex chromosome mutations are better tolerated and more viable compared to changes in autosomes. This is explained by relatively low gene density in both the X and the Y chromosome and by random X chromosome inactivation in mammalian females buffering the effect of X-aneuploidies. However, it is not well understood why apparently similar sex chromosome abnormalities, such as X-monosomy or certain Y chromosome rearrangements, result in different phenotypic effects in different species. It is thought that this is due to species differences in the organization of the Y chromosome, differences in the set of genes escaping X-inactivation, and the presence of species/lineage specific sex-linked genes with functions in development and reproduction. Current knowledge about the species differences in sex chromosome organization and function is limited, this despite the availability of reference genome assemblies for most domestic species. It appears that sequence assembly of the X chromosome in most species is rather patchy containing multiple gaps and possible misassemblies, being the poorest in the pseudoautosomal region and in regions containing putative lineage-specific sequences. The Y chromosome, on the other hand, is typically not included in the reference genome and is studied separately, whereas complete sequence assembly of the male-specific portion of the Y is not yet available for any domestic species. In this talk I will discuss comparative organization and function of animal sex chromosomes and related phenotypes proceeding from our research in horses.


June 1, 2021  |  

Immune regions are no longer incomprehensible with SMRT Sequencing

The complex immune regions of the genome, including MHC and KIR, contain large copy number variants (CNVs), a high density of genes, hyper-polymorphic gene alleles, and conserved extended haplotypes (CEH) with enormous linkage disequilibrium (LDs). This level of complexity and inherent biases of short-read sequencing make it challenging for extracting immune region haplotype information from reference-reliant, shotgun sequencing and GWAS methods. As NGS based genome and exome sequencing and SNP arrays have become a routine for population studies, numerous efforts are being made for developing software to extract and or impute the immune gene information from these datasets. Despite these efforts, the fine mapping of causal variants of immune genes for their well-documented association with cancer, drug-induced hypersensitivity and immune-related diseases, has been slower than expected. This has in many ways limited our understanding of the mechanisms leading to immune disease. In the present work, we demonstrate the advantages of long reads delivered by SMRT Sequencing for assembling complete haplotypes of MHC and KIR gene clusters, as well as calling correct genotypes of genes comprised within them. All the genotype information is detected at allele- level with full phasing information across SNP-poor regions. Genotypes were called correctly from targeted gene amplicons, haplotypes, as well as from a completely assembled 5 Mb contig of the MHC region from a de novo assembly of whole genome shotgun data. De novo analysis pipeline used in all these approaches allowed for reference-free analysis without imputation, a key for interrogation without prior knowledge about ethnic backgrounds. These methods are thus easily adoptable for previously uncharacterized human or non-human species.


June 1, 2021  |  

An improved circular consensus algorithm with an application to detection of HIV-1 Drug-Resistance Associated Mutations (DRAMs)

Scientists who require confident resolution of heterogeneous populations across complex regions have been unable to transition to short-read sequencing methods. They continue to depend on Sanger Sequencing despite its cost and time inefficiencies. Here we present a new redesigned algorithm that allows the generation of circular consensus sequences (CCS) from individual SMRT Sequencing reads. With this new algorithm, dubbed CCS2, it is possible to reach arbitrarily high quality across longer insert lengths at a lower cost and higher throughput than Sanger Sequencing. We apply this new algorithm, dubbed CCS2, to the characterization of the HIV-1 K103N drug-resistance associated mutation, which is both important clinically, and represents a challenge due to regional sequence context. A mutation was introduced into the 3rd position of amino acid position 103 (A>C substitution) of the RT gene on a pNL4-3 backbone by site-directed mutagenesis. Regions spanning ~1,300 bp were PCR amplified from both the non-mutated and mutant (K103N) plasmids, and were sequenced individually and as a 50:50 mixture. Sequencing data were analyzed using the new CCS2 algorithm, which uses a fully-generative probabilistic model of our SMRT Sequencing process to polish consensus sequences to arbitrarily high accuracy. This result, previously demonstrated for multi-molecule consensus sequences with the Quiver algorithm, is made possible by incorporating per-Zero Mode Waveguide (ZMW) characteristics, thus accounting for the intrinsic changes in the sequencing process that are unique to each ZMW. With CCS2, we are able to achieve a per-read empirical quality of QV30 with 19X coverage. This yields ~5000 1.3 kb consensus sequences with a collective empirical quality of ~QV40. Additionally, we demonstrate a 0% miscall rate in both unmixed samples, and estimate a 48:52% frequency for the K103N mutation in the mixed sample, consistent with data produced by orthogonal platforms.


June 1, 2021  |  

An improved circular consensus algorithm with an application to detect HIV-1 Drug Resistance Associated Mutations (DRAMs)

Scientists who require confident resolution of heterogeneous populations across complex regions have been unable to transition to short-read sequencing methods. They continue to depend on Sanger sequencing despite its cost and time inefficiencies. Here we present a new redesigned algorithm that allows the generation of circular consensus sequences (CCS) from individual SMRT Sequencing reads. With this new algorithm, dubbed CCS2, it is possible to reach high quality across longer insert lengths at a lower cost and higher throughput than Sanger sequencing. We applied CCS2 to the characterization of the HIV-1 K103N drug-resistance associated mutation in both clonal and patient samples. This particular DRAM has previously proved to be clinically relevant, but challenging to characterize due to regional sequence context. First, a mutation was introduced into the 3rd position of amino acid position 103 (A>C substitution) of the RT gene on a pNL4-3 backbone by site-directed mutagenesis. Regions spanning ~1.3 kb were PCR amplified from both the non-mutated and mutant (K103N) plasmids, and were sequenced individually and as a 50:50 mixture. Additionally, the proviral reservoir of a subject with known dates of virologic failure of an Efavirenz-based regimen and with documented emergence of drug resistant (K103N) viremia was sequenced at several time points as a proof-of-concept study to determine the kinetics of retention and decay of K103N.Sequencing data were analyzed using the new CCS2 algorithm, which uses a fully-generative probabilistic model of our SMRT Sequencing process to polish consensus sequences to high accuracy. With CCS2, we are able to achieve a per-read empirical quality of QV30 (99.9% accuracy) at 19X coverage. A total of ~5000 1.3 kb consensus sequences with a collective empirical quality of ~QV40 (99.99%) were obtained for each sample. We demonstrate a 0% miscall rate in both unmixed control samples, and estimate a 48:52 frequency for the K103N mutation in the mixed (50:50) plasmid sample, consistent with data produced by orthogonal platforms. Additionally, the K103N escape variant was only detected in proviral samples from time points subsequent (19%) to the emergence of drug resistant viremia. This tool might be used to monitor the HIV reservoir for stable evolutionary changes throughout infection.


June 1, 2021  |  

Best practices for diploid assembly of complex genomes using PacBio: A case study of Cascade Hops

A high quality reference genome is an essential resource for plant and animal breeding and functional and evolutionary studies. The common hop (Humulus lupulus, Cannabaceae) is an economically important crop plant used to flavor and preserve beer. Its genome is large (flow cytometrybased estimates of diploid length >5.4Gb1), highly repetitive, and individual plants display high levels of heterozygosity, which make assembly of an accurate and contiguous reference genome challenging with conventional short-read methods. We present a contig assembly of Cascade Hops using PacBio long reads and the diploid genome assembler, FALCON-Unzip2. The assembly has dramatically improved contiguity and completeness over earlier short-read assemblies. The genome is primarily assembled as haplotypes due to the outbred nature of the organism. We explore patterns of haplotype divergence across the assembly and present strategies to deduplicate haplotypes prior to scaffolding


June 1, 2021  |  

Full-length transcript profiling with the Iso-Seq method for improved genome annotations

Incomplete annotation of genomes represents a major impediment to understanding biological processes, functional differences between species, and evolutionary mechanisms. Often, genes that are large, embedded within duplicated genomic regions, or associated with repeats are difficult to study by short-read expression profiling and assembly. In addition, most genes in eukaryotic organisms produce alternatively spliced isoforms, broadening the diversity of proteins encoded by the genome, which are difficult to resolve with short-read methods. Short-read RNA sequencing (RNA-seq) works by physically shearing transcript isoforms into smaller pieces and bioinformatically reassembling them, leaving opportunity for misassembly or incomplete capture of the full diversity of isoforms from genes of interest. In contrast, Single Molecule, Real-Time (SMRT) Sequencing directly sequences full-length transcripts without the need for assembly and imputation. Here we apply the Iso-Seq method (long-read RNA sequencing) to detect full-length isoforms and the new IsoPhase algorithm to retrieve allele-specific isoform information for two avian models of vocal learning, Anna’s hummingbird (Calypte anna) and zebra finch (Taeniopygia guttata).


June 1, 2021  |  

Comparative metagenome-assembled genome analysis of “Candidatus Lachnocurva vaginae”, formerly known as Bacterial Vaginosis Associated bacterium – 1 (BVAB1)

Bacterial Vaginosis Associated bacterium 1 (BVAB1) is an as-yet uncultured bacterial species found in the human vagina that belongs to the family Lachnospiraceae within the order Clostridiales. As its name suggests, this bacterium is often associated with bacterial vaginosis (BV), a common vaginal disorder that has been shown to increase a woman’s risk for HIV, Chlamydia trachomatis, and Neisseria gonorrhoeae infections as well as preterm birth. Further, BVAB1 is associated with the persistence of BV following metronidazole treatment, increased vaginal inflammation, and adverse obstetrics outcomes. There is no available complete genome sequence of BVAB1, which has made it di?cult to mechanistically understand its role in disease. We present here a circularized metagenome-assembled genome (cMAG) of B VAB1 as well as a comparative analysis including an additional six metagenome-assembled genomes (MAGs) of this species. These sequences were derived from cervicovaginal samples of seven separate women. The cMAG is 1.649 Mb in size and encodes 1,578 genes. We propose to rename BVAB1 to “Candidatus Lachnocurva vaginae” based on phylogenetic analyses, and provide genomic evidence that this candidate species may metabolize D-lactate, produce trimethylamine (one of the chemicals responsible for BV-associated odor), and be motile. The cMAG and the six MAGs are valuable resources that will further contribute to our understanding of the heterogeneous etiology of bacterial vaginosis.


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