June 1, 2021  |  

Profiling metagenomic communities using circular consensus and Single Molecule, Real-Time Sequencing

There are many sequencing-based approaches to understanding complex metagenomic communities, spanning targeted amplification to whole-sample shotgun sequencing. While targeted approaches provide valuable data at low sequencing depth, they are limited by primer design and PCR amplification. Whole-sample shotgun experiments require a high depth of coverage. As such, rare community members may not be represented in the resulting assembly. Circular-consensus, Single Molecule, Real-Time (SMRT) Sequencing reads in the 1-2 kb range, with >99% consensus accuracy, can be efficiently generated for low amounts of input DNA, e.g. as little as 10 ng of input DNA sequenced in 4 SMRT Cells can generate >100,000 such reads. While throughput is low compared to second-generation sequencing, the reads are a true random sampling of the underlying community. Long read lengths translate to a high number of the reads harboring full genes or even full operons for downstream analysis. Here we present the results of circular-consensus sequencing on a mock metagenomic community with an abundance range of multiple orders of magnitude, and compare the results with both 16S and shotgun assembly methods. We show that even with relatively low sequencing depth, the long-read, assembly-free, random sampling allows to elucidate meaningful information from the very low-abundance community members. For example, given the above low-input sequencing approach, a community member at 1/1,000 relative abundance would generate 100 1-2 kb sequence fragments having 99% consensus accuracy, with a high probability of containing a gene fragment useful for taxonomic classification or functional insight.


June 1, 2021  |  

Long-read assembly of the Aedes aegypti Aag2 cell line genome resolves ancient endogenous viral elements

Transmission of arboviruses such as Dengue Virus by Aedes aegypti causes debilitating disease across the globe. Disease in humans can include severe acute symptoms such as hemorrhagic fever and organ failure, but mosquitoes tolerate high titers of virus in a persistent infection. The mechanisms responsible for this viral tolerance are unclear. Recent publications highlighted the integration of genetic material from non-retroviral RNA viruses into the genome of the host during infection that relies upon endogenous retro-transcriptase activity from transposons. These endogenous viral elements (EVEs) found in the genome are predicted to be ancient, and at least some EVEs are under purifying selection, suggesting they are beneficial to the host. To characterize EVE biogenesis in a tractable system, we sequenced the Ae. aegypti cell line, Aag2, to 58-fold coverage and present a de novo assembly of the genome. The assembly contains 1.7 Gb of genomic and 255 Mb of alternative haplotype specific sequence, consisting of contigs with a N50 of 1.4 Mb; a value that, when compared with other assemblies of the Aedes genus, is from 1-3 orders of magnitude longer. The Aag2 genome is highly repetitive (70%), most of which is classified as transposable elements (60%). We identify EVEs in the genome homologous to a range of extant viruses, many of which cluster in these regions of repetitive DNA. The contiguous assembly allows for more comprehensive identification of the transposable elements and EVEs that are most likely to be lost in assemblies lacking the read length of SMRT Sequencing.


June 1, 2021  |  

An update on goat genomics

Goats are specialized in dairy, meat and fiber production, being adapted to a wide range of environmental conditions and having a large economic impact in developing countries. In the last years, there have been dramatic advances in the knowledge of the structure and diversity of the goat genome/transcriptome and in the development of genomic tools, rapidly narrowing the gap between goat and related species such as cattle and sheep. Major advances are: 1) publication of a de novo goat genome reference sequence; 2) Development of whole genome high density RH maps, and; 3) Design of a commercial 50K SNP array. Moreover, there are currently several projects aiming at improving current genomic tools and resources. An improved assembly of the goat genome using PacBio reads is being produced, and the design of new SNP arrays is being studied to accommodate the specific needs of this species in the context of very large scale genotyping projects (i.e. breed characterization at an international scale and genomic selection) and parentage analysis. As in other species, the focus has now turned to the identification of causative mutations underlying the phenotypic variation of traits. In addition, since 2014, the ADAPTmap project (www.goatadaptmap.org) has gathered data to explore the diversity of caprine populations at a worldwide scale by using a wide variety of approaches and data.


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  |  

From Sequencing to Chromosomes: New de novo assembly and scaffolding methods improve the goat reference genome

Single-molecule sequencing is now routinely used to assemble complete, high-quality microbial genomes, but these assembly methods have not scaled well to large genomes. To address this problem, we previously introduced the MinHash Alignment Process (MHAP) for overlapping single-molecule reads using probabilistic, locality-sensitive hashing. Integrating MHAP with Celera Assembler (CA) has enabled reference-grade assemblies of model organisms, revealing novel heterochromatic sequences and filling low-complexity gap sequences in the GRCh38 human reference genome. We have applied our methods to assemble the San Clemente goat genome. Combining single-molecule sequencing from Pacific Biosciences and BioNano Genomics generates and assembly that is over 150-fold more contiguous than the latest Capra hircus reference. In combination with Hi-C sequencing, the assembly surpasses reference assemblies, de novo, with minimal manual intervention. The autosomes are each assembled into a single scaffold. Our assembly provides a more complete gene reconstruction, better alignments with Goat 52k chip, and improved allosome reconstruction. In addition to providing increased continuity of sequence, our assembly achieves a higher BUSCO completion score (84%) than the existing goat reference assembly suggesting better quality annotation of gene models. Our results demonstrate that single-molecule sequencing can produce near-complete eukaryotic genomes at modest cost and minimal manual effort.


June 1, 2021  |  

Profiling the microbiome in fecal microbiota transplantation using circular consensus and Single Molecule, Real-Time Sequencing

There are many sequencing-based approaches to understanding complex metagenomic communities spanning targeted amplification to whole-sample shotgun sequencing. While targeted approaches provide valuable data at low sequencing depth, they are limited by primer design and PCR. Whole-sample shotgun experiments generally use short-read sequencing, which results in data processing difficulties. For example, reads less than 500bp in length will rarely cover a complete gene or region of interest, and will require assembly. This not only introduces the possibility of incorrectly combining sequence from different community members, it requires a high depth of coverage. As such, rare community members may not be represented in the resulting assembly. Circular-consensus, single molecule, real-time (SMRT®) Sequencing reads in the 1-3kb range, with >99% accuracy can be efficiently generated for low amounts of input DNA. 10 ng of input DNA sequenced in 4 SMRT Cells on the PacBio RS II would generate >100,000 such reads. While throughput is lower compared to short-read sequencing methods, the reads are a true random sampling of the underlying community since SMRT Sequencing has been shown to have very low sequence-context bias. With reads >1 kb at >99% accuracy it is reasonable to expect a high percentage of reads include gene fragments useful for analysis without the need for de novo assembly. Here we present the results of circular consensus sequencing for an individual’s microbiome, before and after undergoing fecal microbiota transplantation (FMT) in order to treat a chronic Clostridium difficile infection. We show that even with relatively low sequencing depth, the long-read, assembly-free, random sampling allows us to profile low abundance community members at the species level. We also show that using shotgun sampling with long reads allows a level of functional insight not possible with classic targeted 16S, or short read sequencing, due to entire genes being covered in single reads.


June 1, 2021  |  

Long-read assembly of the Aedes aegypti Aag2 cell line genome resolves ancient endogenous viral elements

Transmission of arboviruses such as Dengue and Zika viruses by Aedes aegypti causes widespread and debilitating disease across the globe. Disease in humans can include severe acute symptoms such as hemorrhagic fever, organ failure, and encephalitis; and yet, mosquitoes tolerate high titers of virus in a persistent infection. The mechanisms responsible for tolerance to viral infection in mosquitoes are still unclear. Recent publications have highlighted the integration of genetic material from non-retroviral RNA viruses into the genome of the host during infection that relies upon endogenous retro-transcriptase activity from transposons. These endogenous viral elements (EVEs) found in the genome are predicted to be ancient and at least some EVEs are under purifying selection, which suggests that they are beneficial to the host. In order characterize EVE biogenesis in a tractable system we sequenced the Ae. aegypti cell line, Aag2, to 58X coverage and here present a de novo assembly of the genome. The assembly consists of 1.7 Gb of genomic and 255 Mb of alternative haplotype specific sequence, made up of contigs with a N50 of 1.4 Mb; a value that, when compared with other assemblies of the Aedes genus, is from 1-3 orders of magnitude longer. The Aag2 genome is highly repetitive (70%), most of which is classified as transposable elements (60%). We identify a plethora of EVEs in the genome homologous to a diverse range of extant viruses, many of which cluster in these regions of highly repetitive DNA. The highly contiguous nature of this assembly allows for a more comprehensive identification of the transposable elements and EVEs that are most likely to be lost in assemblies lacking the read length of SMRT Sequencing. Transmission of arboviruses such as Dengue Virus by Aedes aegypti causes widespread and debilitating disease across the globe. Disease in humans can include severe acute symptoms such as hemorrhagic fever, organ failure, and encephalitis; and yet, mosquitoes tolerate high titers of virus in a persistent infection. The mechanisms responsible for tolerance to viral infection in mosquitoes are still unclear. Recent publications have highlighted the integration of genetic material from non-retroviral RNA viruses into the genome of the host during infection that relies upon endogenous retro-transcriptase activity from transposons. These endogenous viral elements (EVEs) found in the genome are predicted to be ancient and at least some EVEs are under purifying selection, which suggests that they are beneficial to the host. In order characterize EVE biogenesis in a tractable system we sequenced the Ae. aegypti cell line, Aag2, to 58X coverage and here present a de novo assembly of the genome. The assembly consists of 1.7 Gb of genomic and 255 Mb of alternative haplotype specific sequence, made up of contigs with a N50 of 1.4 Mb; a value that, when compared with other assemblies of the Aedes genus, is from 1-3 orders of magnitude longer. The Aag2 genome is highly repetitive (70%), most of which is classified as transposable elements (60%). We identify a plethora of EVEs in the genome homologous to a diverse range of extant viruses, many of which cluster in these regions of highly repetitive DNA. The highly contiguous nature of this assembly allows for a more comprehensive identification of the transposable elements and EVEs that are most likely to be lost in assemblies lacking the read length of SMRT Sequencing. Transmission of arboviruses such as Dengue Virus by Aedes aegypti causes widespread and debilitating disease across the globe. Disease in humans can include severe acute symptoms such as hemorrhagic fever, organ failure, and encephalitis; and yet, mosquitoes tolerate high titers of virus in a persistent infection. The mechanisms responsible for tolerance to viral infection in mosquitoes are still unclear.


June 1, 2021  |  

Workflow for processing high-throughput, Single Molecule, Real-Time Sequencing data for analyzing the microbiome of patients undergoing fecal microbiota transplantation

There are many sequencing-based approaches to understanding complex metagenomic communities spanning targeted amplification to whole-sample shotgun sequencing. While targeted approaches provide valuable data at low sequencing depth, they are limited by primer design and PCR. Whole-sample shotgun experiments generally use short-read sequencing, which results in data processing difficulties. For example, reads less than 500 bp in length will rarely cover a complete gene or region of interest, and will require assembly. This not only introduces the possibility of incorrectly combining sequence from different community members, it requires a high depth of coverage. As such, rare community members may not be represented in the resulting assembly. Circular-consensus, Single Molecule, Real-Time (SMRT) Sequencing reads in the 1-3 kb range, with >99% accuracy can be generated using the previous generation PacBio RS II or, in much higher throughput, using the new Sequel System. While throughput is lower compared to short-read sequencing methods, the reads are a true random sampling of the underlying community since SMRT Sequencing has been shown to have very low sequence-context bias. With single-molecule reads >1 kb at >99% consensus accuracy, it is reasonable to expect a high percentage of reads to include genes or gene fragments useful for analysis without the need for de novo assembly. Here we present the results of circular consensus sequencing for an individual’s microbiome, before and after undergoing fecal microbiota transplantation (FMT) in order to treat a chronic Clostridium difficile infection. We show that even with relatively low sequencing depth, the long-read, assembly-free, random sampling allows us to profile low abundance community members at the species level. We also show that using shotgun sampling with long reads allows a level of functional insight not possible with classic targeted 16S, or short read sequencing, due to entire genes being covered in single reads.


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.


June 1, 2021  |  

De novo assembly and preliminary annotation of the Schizocardium californicum genome

Animals in the phylum Hemichordata have provided key understanding of the origins and development of body patterning and nervous system organization. However, efforts to sequence and assemble the genomes of highly heterozygous non-model organisms have proven to be difficult with traditional short read approaches. Long repetitive DNA structures, extensive structural variation between haplotypes in polyploid species, and large genome sizes are limiting factors to achieving highly contiguous genome assemblies. Here we present the highly contiguous de novo assembly and preliminary annotation of an indirect developing hemichordate genome, Schizocardium californicum, using SMRT Sequening long reads.


June 1, 2021  |  

Unbiased characterization of metagenome composition and function using HiFi sequencing on the PacBio Sequel II System

Recent work comparing metagenomic sequencing methods indicates that a comprehensive picture of the taxonomic and functional diversity of complex communities will be difficult to achieve with short-read technology alone. While the lower cost of short reads has enabled greater sequencing depth, the greater contiguity of long-read assemblies and lack of GC bias in SMRT Sequencing has enabled better gene finding. However, since long-read assembly requires high coverage for error correction, the benefits of unbiased coverage have in the past been lost for low abundance species. SMRT Sequencing performance improvements and the introduction of the Sequel II System has enabled a new, high throughput data type uniquely suited to metagenome characterization: HiFi reads. HiFi reads combine high accuracy with read lengths up to 15 kb, eliminating the need for assembly for most microbiome applications, including functional profiling, gene discovery, and metabolic pathway reconstruction. Here we present the application of the HiFi data type to enable a new method of analyzing metagenomes that does not require assembly.


June 1, 2021  |  

High-quality human genomes achieved through HiFi sequence data and FALCON-Unzip assembly

De novo assemblies of human genomes from accurate (85-90%), continuous long reads (CLR) now approach the human reference genome in contiguity, but the assembly base pair accuracy is typically below QV40 (99.99%), an order-of-magnitude lower than the standard for finished references. The base pair errors complicate downstream interpretation, particularly false positive indels that lead to false gene loss through frameshifts. PacBio HiFi sequence data, which are both long (>10 kb) and very accurate (>99.9%) at the individual sequence read level, enable a new paradigm in human genome assembly. Haploid human assemblies using HiFi data achieve similar contiguity to those using CLR data and are highly accurate at the base level1. Furthermore, HiFi assemblies resolve more high-identity sequences such as segmental duplications2. To enable HiFi assembly in diploid human samples, we have extended the FALCON-Unzip assembler to work directly with HiFi reads. Here we present phased human diploid genome assemblies from HiFi sequencing of HG002, HG005, and the Vertebrate Genome Project (VGP) mHomSap1 trio on the PacBio Sequel II System. The HiFi assemblies all exceed the VGP’s quality guidelines, approaching QV50 (99.999%) accuracy. For HG002, 60% of the genome was haplotype-resolved, with phase-block N50 of 143Kbp and phasing accuracy of 99.6%. The overall mean base accuracy of the assembly was QV49.7. In conclusion, HiFi data show great promise towards complete, contiguous, and accurate diploid human assemblies.


June 1, 2021  |  

Unbiased characterization of metagenome composition and function using HiFi sequencing on the PacBio Sequel II System

Recent work comparing metagenomic sequencing methods indicates that a comprehensive picture of the taxonomic and functional diversity of complex communities will be difficult to achieve with one sequencing technology alone. While the lower cost of short reads has enabled greater sequencing depth, the greater contiguity of long-read assemblies and lack of GC bias in SMRT Sequencing has enabled better gene finding. However, since long-read assembly typically requires high coverage for error correction, these benefits have in the past been lost for low-abundance species. The introduction of the Sequel II System has enabled a new, higher throughput, assembly-optional data type that addresses these challenges: HiFi reads. HiFi reads combine QV20 accuracy with long read lengths, eliminating the need for assembly for most metagenome applications, including gene discovery and metabolic pathway reconstruction. In fact, the read lengths and accuracy of HiFi data match or outperform the quality metrics of most metagenome assemblies, enabling cost-effective recovery of intact genes and operons while omitting the resource intensive and data-inefficient assembly step. Here we present the application of HiFi sequencing to both mock and human fecal samples using full-length 16S and shotgun methods. This proof-of-concept work demonstrates the unique strengths of the HiFi method. First, the high correspondence between the expected community composition,16S and shotgun profiling data reflects low context bias. In addition, every HiFi read yields ~5-8 predicted genes, without assembly, using standard tools. If assembly is desired, excellent results can be achieved with Canu and contig binning tools. In summary, HiFi sequencing is a new, cost-effective option for high-resolution functional profiling of metagenomes which complements existing short read workflows.


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.


June 1, 2021  |  

Improving long-read assembly of microbial genomes and plasmids

Complete, high-quality microbial genomes are very valuable across a broad array of fields, from environmental studies, to human microbiome health, food pathogen surveillance, etc. Long-read sequencing enables accurate resolution of complex microbial genomes and is becoming the new standard. Here we report our novel Microbial Assembly pipeline to facilitate rapid, large-scale analysis of microbial genomes. We sequenced a 48-plex library with one SMRT Cell 8M on the Sequel II System, demultiplexed, then analyzed the data with Microbial Assembly.


Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.