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June 1, 2021  |  

Genome sequencing of endosymbiotic bacterial Streptomyces sp. from Antartic lichen using Single Molecule Real-time Sequencing (SMRT) technology.

Along with the advent of next-generation sequencing (NGS) techniques, it has become possible to sequence a microbial genome very quickly with high coverage. Recently, PacificBioscience developed single molecule real-time sequencing (SMRT) technology, 3rd generation sequencing platform, which provide much longer (average read length: 1.5Kb) reads without PCR amplification. We did de novo sequencing of Streptomyces sp. using Illumina GAIIx, Roche 454 and PacBio RS system and compared the data. The endosymbiotic bacteria Streptomyces sp. PAMC 26508 was isolated from Antarctic lichen Psoroma sp. that grows attached rocks on Barton Peninsula, King George Island, Antarctica (62, 13’S, 58, 47’W). With 4 SMRT cells, we could get more than 15x coverage of corrected sequence data for de novo assembly. Comparing the performance of other sequencing platforms, PacBio platform could generate data on similar manner with general mid-level GC content organism. In conclusion, PacBio RS system, SMRT technology, shows better performance with high GC content organisms and is expected to be the new tool to improve the de novo sequencing and assembly.

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June 1, 2021  |  

Genome sequencing of microbial genomes using Single Molecule Real-time sequencing (SMRT) technology.

In the last year, high-throughput sequencing technologies have progressed from proof-of-concept to production quality. Although each technology is able to produce vast quantities of sequence information, in every case the underlying chemistry limits reads to very short lengths. We present a examining de novo assembly comparison with bacterial genome assembly varying genome size (from 3.1Mb to 7.6Mb) and different G+C contents (from 43% to 71%), respectively. We analyzed Solexa reads, 454 reads and Pacbio RS reads from Streptomyces sp. (Genome size, 7.6 Mb; G+C content, 71%), Psychrobacter sp. (Genome size, 3.5 Mb; G+C content, 43%), Salinibacterium sp. (Genome size, 3.1 Mb; G+C content, 61%) and Frigoribacterium sp. (Genome size, 3.3 Mb; G+C content, 63%). We assembly each bacterial genome using Celera assembler 7.0 with and without PacBio RS reads. We found out that the assemble result with Pacbio RS reads have less contigs and scaffolds, and better N50 values.

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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  |  

Rapid full-length Iso-Seq cDNA sequencing of rice mRNA to facilitate annotation and identify splice-site variation.

PacBio’s new Iso-Seq technology allows for rapid generation of full-length cDNA sequences without the need for assembly steps. The technology was tested on leaf mRNA from two model O. sativa ssp. indica cultivars – Minghui 63 and Zhenshan 97. Even though each transcriptome was not exhaustively sequenced, several thousand isoforms described genes over a wide size range, most of which are not present in any currently available FL cDNA collection. In addition, the lack of an assembly requirement provides direct and immediate access to complete mRNA sequences and rapid unraveling of biological novelties.

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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.

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June 1, 2021  |  

Comparative genome analysis of Clavibacter michiganensis subsp. michiganensis strains provides insights into genetic diversity and virulence.

Clavibacter michiganensis subsp. michiganensis (Cmm) is a gram positive actinomycete, causing bacterial canker of tomato (Solanum lycopersicum) a disease that can cause significant losses in tomato production. In this study, we determined the complete genome sequence of 13 California Cmm strains and one saprophytic Clavibacter strain using a combination of Ilumina and PacBio sequencing. The California Cmm strains have genome size (3.2 -3.3 mb) similar to the reference strain NCPPB382 (3.3 mb) with =98% sequence identity. Cmm strains from California share =92% genes (8-10% are noble genes) with the reference Cmm strain NCPPB382. Despite this similarity, we detected significant alternatives in California strains with respect to plasmid number, plasmid composition, and genomic island presence indicating acquisition of unique mechanisms controlling virulence. Plasmids pCM1 and pCM2, that were previously demonstrated to be required for NCPPB382 virulence, also differ in their presence and gene content across Cmm strains. pCM2 is absent in some Cmm strains and that still retain virulence in tomato. Saprophytic Clavibacter possess a novel plasmid, pSCM, and lacks the majority of characterized virulence factors. Genome sequence information was also used to design specific and sensitive primer pairs for Cmm detection. A mechanistic understanding of how genomic changes have impacted Cmm virulence and survival across diverse strains will be necessary for developing a robust disease control strategies for bacterial canker of tomato.

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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  |  

HLA variant identification techniques

The Human Leukocyte Antigen (HLA) genes located on chromosome 6 are responsible for regulating immune function via antigen presentation and are one of the determining factors for stem cell and organ transplantation compatibility. Additionally various alleles within this region have been implicated in autoimmune disorders, cancer, vaccine response and both non-infectious and infectious disease risk. The HLA region is highly variable; containing repetitive regions; and co-dominantly expressed genes. This complicates short read mapping and means that assessing the effect of variation within a gene requires full phase information to resolve haplotypes.One solution to the problem of HLA identification is the use of statistical inference to suggest the most likely diploid alleles given the genotypes observed. The assumption of this approach is the availability of an extensive reference panel. Whilst there exists good population genetics data for imputing European populations, there remains a paucity of information about variation in African populations. Filling this gap is one of the aims of the Genome Diversity in Africa Project and as a first step we are performing a pilot study to identify the optimal method for determining HLA type information for large numbers of samples from African populations.To that end we have obtained samples from 125 consented African participants selected from 5 populations across Africa (Morrocan, Ashanti, Igbo, Kalenjin, and Zulu). The methods included in our pilot study are Sanger sequencing (ABI), NGS on HiSeqX Ten platform (Illumina); long-range PCR combined with single molecule real-time (SMRT) sequencing (PacBio); and for a subset of samples library preparation on GemCode Platform (10x Genomics), which delivers valuable long range contextual information, combined with Illumina NGS sequencing.Results from capillary sequencing suggests the presence of a minimum of two novel alleles. Long Range PCR have been performed initially on a subset of samples using both primers sourced from GenDX and designed as described in Shiina et al (2012). Initial results from both primer sets were promising on Promega DNA test samples but only the GenDX primers proved effective on the African samples, producing consistently PCR products of the expected size in the Igbo, Ashanti, Morrocan and Zulu samples. We will present early results from our evaluation of the different sequencing technologies

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June 1, 2021  |  

A comprehensive lincRNA analysis: From conifers to trees

We have produced an updated annotation of the Norway spruce genome on the basis of an in siliconormalised set of RNA-Seq data obtained from 1,529 samples and comprising 15.5 billion paired-end Illumina HiSeq reads complemented by 18Mbp of PacBio cDNA data (3.2M sequences). In addition to augmenting and refining the previous protein coding gene annotation, here we focus on the addition of long intergenic non-coding RNA (lincRNA) and micro RNA (miRNA) genes. In addition to non-coding loci, our analyses also identified protein coding genes that had been missed by the initial genome annotation and enabled us to update the annotation of existing gene models. In particular, splice variant information, as supported by PacBio sequencing reads, has been added to the current annotation and previously fragmented gene models have been merged by scaffolding disjoint genomic scaffolds on the basis of transcript evidence. Using this refined annotation, a targeted analysis of the lincRNAs enabled their classification as i) deeply conserved, ii) conserved in seed plants iii) gymnosperm/conifer specific. Concurrently, complementary analyses were performed as part of the aspen genome project and the results of a comparative analysis of the lincRNAs conserved in both Norway spruce and Eurasian aspen enabled us to identify conserved and diverged expression profiles. At present, we are delving further into the expression results with the aim to functionally annotate the lincRNA genes, by developing a co-expression network analyses based GO annotation.

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June 1, 2021  |  

The MHC Diversity in Africa Project (MDAP) pilot – 125 African high resolution HLA types from 5 populations

The major histocompatibility complex (MHC), or human leukocyte antigen (HLA) in humans, is a highly diverse gene family with a key role in immune response to disease; and has been implicated in auto-immune disease, cancer, infectious disease susceptibility, and vaccine response. It has clinical importance in the field of solid organ and bone marrow transplantation, where donors and recipient matching of HLA types is key to transplanted organ outcomes. The Sanger based typing (SBT) methods currently used in clinical practice do not capture the full diversity across this region, and require specific reference sequences to deconvolute ambiguity in HLA types. However, reference databases are based largely on European populations, and the full extent of diversity in Africa remains poorly understood. Here, we present the first systematic characterisation of HLA diversity within Africa in the pilot phase of the MHC Diversity in Africa Project, together with an evaluation of methods to carry out scalable cost-effective, as well as reliable, typing of this region in African populations.To sample a geographically representative panel of African populations we obtained 125 samples, 25 each from the Zulu (South Africa), Igbo (Nigeria), Kalenjin (Kenya), Moroccan and Ashanti (Ghana) groups. For methods validation we included two controls from the International Histocompatibility Working Group (IHWG) collection with known typing information. Sanger typing and Illumina HiSeq X sequencing of these samples indicated potentially novel Class I and Class II alleles; however, we found poor correlation between HiSeq X sequencing and SBT for both classes. Long Range PCR and high resolution PacBio RS-II typing of 4 of these samples identified 7 novel Class II alleles, highlighting the high levels of diversity in these populations, and the need for long read sequencing approaches to characterise this comprehensively. We have now expanded this approach to the entire pilot set of 125 samples. We present these confirmed types and discuss a workflow for scaling this to 5000 individuals across Africa.The large number of new alleles identified in our pilot suggests the high level of African HLA diversity and the utility of high resolution methods. The MDAP project will provide a framework for accurate HLA typing, in addition to providing an invaluable resource for imputation in GWAS, boosting power to identify and resolve HLA disease associations.

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June 1, 2021  |  

Assessing diversity and clonal variation of Australia’s grapevine germplasm: Curating the FALCON-Unzip Chardonnay de novo genome assembly

Until recently only two genome assemblies were publicly available for grapevine—both Vitis vinifera L. Cv. Pinot Noir (PN). The best available PN genome assembly (Jaillon et al. 2007) is not representative of the genome complexity that is typical of wine-grape cultivars in the field and it is highly fragmented. To assess the genetic complexities of Chardonnay grapevine, assembly of a new de novo reference genome was needed. Here we describe a draft assembly using PacBio SMRT Sequencing data and PacBio’s new phased diploid genome assembler FALCON-Unzip (Chin et al. 2016).

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June 1, 2021  |  

Profiling complex population genomes with highly accurate single molecule reads: cow rumen microbiomes

Determining compositions and functional capabilities of complex populations is often challenging, especially for sequencing technologies with short reads that do not uniquely identify organisms or genes. Long-read sequencing improves the resolution of these mixed communities, but adoption for this application has been limited due to concerns about throughput, cost and accuracy. The recently introduced PacBio Sequel System generates hundreds of thousands of long and highly accurate single-molecule reads per SMRT Cell. We investigated how the Sequel System might increase understanding of metagenomic communities. In the past, focus was largely on taxonomic classification with 16S rRNA sequencing. Recent expansion to WGS sequencing enables functional profiling as well, with the ultimate goal of complete genome assemblies. Here we compare the complex microbiomes in 5 cow rumen samples, for which Illumina WGS sequence data was also available. To maximize the PacBio single-molecule sequence accuracy, libraries of 2 to 3 kb were generated, allowing many polymerase passes per molecule. The resulting reads were filtered at predicted single-molecule accuracy levels up to 99.99%. Community compositions of the 5 samples were compared with Illumina WGS assemblies from the same set of samples, indicating rare organisms were often missed with Illumina. Assembly from PacBio CCS reads yielded a contig >100 kb in length with 6-fold coverage. Mapping of Illumina reads to the 101 kb contig verified the PacBio assembly and contig sequence. These results illustrate ways in which long accurate reads benefit analysis of complex communities.

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June 1, 2021  |  

High-quality, highly contiguous re-assembly of the pig genome

Many applications of high throughput sequencing rely on the availability of an accurate reference genome. Errors in the reference genome assembly increase the number of false-positives in downstream analyses. Recently, we have shown that over 33% of the current pig reference genome, Sscrofa10.2, is either misassembled or otherwise unreliable for genomic analyses. Additionally, ~10% of the bases in the assembly are Ns in gaps of an arbitrary size. Thousands of highly fragmented contigs remain unplaced and many genes are known to be missing from the assembly. Here we present a new assembly of the pig genome, Sscrofa11, assembled using 65X PacBio sequencing from T.J. Tabasco, the same Duroc sow used in the assembly of Sscrofa10.2. The PacBio reads were assembled using the Falcon assembly pipeline resulting in 3,206 contigs with an initial contig N50 of 14.5Mb. We used Sscrofa10.2 as a template to scaffold the PacBio contigs, under the assumption that its gross structure is correct, and used PBJelly to fill gaps. Additional gaps were filled using large, sequenced BACs from the original assembly. Following gap filling, the assembly has substantially improved contiguity and contains more sequence than the Sscrofa10.2 assembly. Arrow and Pilon were used to polish the assembly. The contig N50 is now 58.5Mb with 103 gaps remaining. By comparing regions of the two assemblies we show that regions with structural abnormalities we identified in Sscrofa10.2 are resolved in the new PacBio assembly.

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June 1, 2021  |  

Profiling complex communities with highly accurate single molecule reads: cow rumen microbiomes

Determining compositions and functional capabilities of complex populations is often challenging, especially for sequencing technologies with short reads that do not uniquely identify organisms or genes. Long-read sequencing improves the resolution of these mixed communities, but adoption for this application has been limited due to concerns about throughput, cost and accuracy. The recently introduced PacBio Sequel System generates hundreds of thousands of long and highly accurate single-molecule reads per SMRT Cell. We investigated how the Sequel System might increase understanding of metagenomic communities. In the past, focus was largely on taxonomic classification with 16S rRNA sequencing. Recent expansion to WGS sequencing enables functional profiling as well, with the ultimate goal of complete genome assemblies. Here we compare the complex microbiomes in 5 cow rumen samples, for which Illumina WGS sequence data was also available. To maximize the PacBio single-molecule sequence accuracy, libraries of 2 to 3 kb were generated, allowing many polymerase passes per molecule. The resulting reads were filtered at predicted single-molecule accuracy levels up to 99.99%. Community compositions of the 5 samples were compared with Illumina WGS assemblies from the same set of samples, indicating rare organisms were often missed with Illumina. Assembly from PacBio CCS reads yielded a contig >100 kb in length with 6-fold coverage. Mapping of Illumina reads to the 101 kb contig verified the PacBio assembly and contig sequence. Scaffolding with reads from a PacBio unsheared library produced a complete genome of 2.4 Mb. These results illustrate ways in which long accurate reads benefit analysis of complex communities.

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June 1, 2021  |  

Amplification-free, CRISPR-Cas9 targeted enrichment and SMRT Sequencing of repeat-expansion disease causative genomic regions

Targeted sequencing has proven to be economical for obtaining sequence information for defined regions of the genome. However, most target enrichment methods are reliant upon some form of amplification which can negatively impact downstream analysis. For example, amplification removes epigenetic marks present in native DNA, including nucleotide methylation, which are hypothesized to contribute to disease mechanisms in some disorders. In addition, some genomic regions known to be causative of many genetic disorders have extreme GC content and/or repetitive sequences that tend to be recalcitrant to faithful amplification. We have developed a novel, amplification-free enrichment technique that employs the CRISPR/Cas9 system to target individual genes. This method, in conjunction with the long reads, high consensus accuracy, and uniform coverage of SMRT Sequencing, allows accurate sequence analysis of complex genomic regions that cannot be investigated with other technologies. Using this strategy, we have successfully targeted a number of repeat expansion disorder loci (HTT, FMR1, ATXN10, C9orf72).With this data, we demonstrate the ability to isolate thousands of individual on-target molecules and, using the Sequel System, accurately sequence through long repeats regardless of the extreme GC-content. The method is compatible with multiplexing of multiple target loci and multiple samples in a single reaction. Furthermore, because there is no amplification step, this technique also preserves native DNA molecules for sequencing, allowing for the direct detection and characterization of epigenetic signatures. To this end, we demonstrate the detection of 5-mC in the CGG repeat of the FMR1 gene that is responsible for Fragile X syndrome.

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