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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).

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June 1, 2021

Best practices for whole genome sequencing using the Sequel System

Plant and animal whole genome sequencing has proven to be challenging, particularly due to genome size, high density of repetitive elements and heterozygosity. The Sequel System delivers long reads, high consensus accuracy and uniform coverage, enabling more complete, accurate, and contiguous assemblies of these large complex genomes. The latest Sequel chemistry increases yield up to 8 Gb per SMRT Cell for long insert libraries >20 kb and up to 10 Gb per SMRT Cell for libraries >40 kb. In addition, the recently released SMRTbell Express Template Prep Kit reduces the time (~3 hours) and DNA input (~3 µg), making the workflow easy to use for multi- SMRT Cell projects. Here, we recommend the best practices for whole genome sequencing and de novo assembly of complex plant and animal genomes. Guidelines for constructing large-insert SMRTbell libraries (>30 kb) to generate optimal read lengths and yields using the latest Sequel chemistry are presented. We also describe ways to maximize library yield per preparation from as littles as 3 µg of sheared genomic DNA. The combination of these advances makes plant and animal whole genome sequencing a practical application of the Sequel System.

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June 1, 2021

Haplotyping using full-length transcript sequencing reveals allele-specific expression

An important need in analyzing complex genomes is the ability to separate and phase haplotypes. While whole genome assembly can deliver this information, it cannot reveal whether there is allele-specific gene or isoform expression. The PacBio Iso-Seq method, which can produce high-quality transcript sequences of 10 kb and longer, has been used to annotate many important plant and animal genomes. We present an algorithm called IsoPhase that post-processes Iso-Seq data for transcript-based haplotyping. We applied IsoPhase to a maize Iso-Seq dataset consisting of two homozygous parents and two F1 cross hybrids. We validated the majority of the SNPs called with IsoPhase against matching short read data and identified cases of allele-specific, gene-level and isoform-level expression.

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June 1, 2021

A low DNA input protocol for high-quality PacBio de novo genome assemblies from single invertebrate individuals

A high-quality reference genome is an essential tool for studies of plant and animal genomics. PacBio Single Molecule, Real-Time (SMRT) Sequencing generates long reads with uniform coverage and high consensus accuracy, making it a powerful technology for de novo genome assembly. PacBio is the core technology for many large genome initiatives, however, relatively high DNA input requirements (5 µg for standard library protocol) have placed PacBio out of reach for many projects on small, non-inbred organisms that may have lower DNA content. Here we present high-quality de novo genome assemblies from single invertebrate individuals for two different species: the Anopheles coluzzii mosquito and the Schistosoma mansoni parasitic flatworm. A modified SMRTbell library construction protocol without DNA shearing and size selection was used to generate a SMRTbell library from just 50-100 ng of starting genomic DNA. The libraries were run on the Sequel System with chemistry v3.0 and software v6.0, generating a range of 21-32 Gb of sequence per SMRT Cell with 20 hour movies, and followed by diploid de novo genome assembly with FALCON-Unzip. The resulting assemblies had high contiguity (contig N50s over 3 Mb for both species) and completeness (as determined by conserved BUSCO gene analysis). We were also able to resolve maternal and paternal haplotypes for 1/3 of the genome in both cases. By sequencing and assembling material from a single diploid individual, only two haplotypes are present, simplifying the assembly process compared to samples from multiple pooled individuals. This new low-input approach puts PacBio-based assemblies in reach for small, highly heterozygous organisms that comprise much of the diversity of life. The method presented here can be applied to samples with starting DNA amounts around 100 ng per 250 Mb – 1 Gb genome size.

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June 1, 2021

Library prep and bioinformatics improvements for full-length transcript sequencing on the PacBio Sequel System

The PacBio Iso-Seq method produces high-quality, full-length transcripts of up to 10 kb and longer and has been used to annotate many important plant and animal genomes. Here we describe an improved, simplified library workflow and analysis pipeline that reduces library preparation time, RNA input, and cost. The Iso-Seq V2 Express workflow is a one day protocol that requires only ~300 ng of total RNA input while also reducing the number of reverse transcription and amplification steps down to single reactions. Compared with the previous workflow, the Iso-Seq V2 Express workflow increases the percentage of full-length (FL) reads while achieving a higher average transcript length. At the same time, the Iso-Seq 3 analysis recently released in the SMRT Link 6.0 software is a major improvement over previous versions. Iso-Seq 3 is highly accurate at detecting and removing library artifacts (TSO and RT artifacts) as well as differentiating barcodes on multiplexed samples. Iso-Seq 3 achieves the same output performance in high-quality transcript sequences compared to previous versions while reducing the runtime and memory usage dramatically.

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June 1, 2021

Single molecule high-fidelity (HiFi) Sequencing with >10 kb libraries

Recent improvements in sequencing chemistry and instrument performance combine to create a new PacBio data type, Single Molecule High-Fidelity reads (HiFi reads). Increased read length and improvement in library construction enables average read lengths of 10-20 kb with average sequence identity greater than 99% from raw single molecule reads. The resulting reads have the accuracy comparable to short read NGS but with 50-100 times longer read length. Here we benchmark the performance of this data type by sequencing and genotyping the Genome in a Bottle (GIAB) HG0002 human reference sample from the National Institute of Standards and Technology (NIST). We further demonstrate the general utility of HiFi reads by analyzing multiple clones of Cabernet Sauvignon. Three different clones were sequenced and de novo assembled with the CANU assembly algorithm, generating draft assemblies of very high contiguity equal to or better than earlier assembly efforts using PacBio long reads. Using the Cabernet Sauvignon Clone 8 assembly as a reference, we mapped the HiFi reads generated from Clone 6 and Clone 47 to identify single nucleotide polymorphisms (SNPs) and structural variants (SVs) that are specific to each of the three samples.

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June 1, 2021

A high-quality de novo genome assembly from a single mosquito using PacBio sequencing

A high-quality reference genome is an essential tool for studies of plant and animal genomics. PacBio Single Molecule, Real-Time (SMRT) Sequencing generates long reads with uniform coverage and high consensus accuracy, making it a powerful technology for de novo genome assembly. While PacBio is the core technology for many large genome initiatives, relatively high DNA input requirements (3 µg for standard library protocol) have placed PacBio out of reach for many projects on small, non-inbred organisms that may have lower DNA content. Here we present high-quality de novo genome assemblies from single invertebrate individuals for two different species: the Anopheles coluzzii mosquito and the Schistosoma mansoni parasitic flatworm. A modified SMRTbell library construction protocol without DNA shearing and size selection was used to generate a SMRTbell library from just 150 ng of starting genomic DNA. The libraries were run on the Sequel System with chemistry v3.0 and software v6.0, generating a range of 21-32 Gb of sequence per SMRT Cell with 20-hour movies (10-12 Gb for 10-hour movies), and followed by diploid de novo genome assembly with FALCON-Unzip. The resulting assemblies had high contiguity (contig N50s over 3 Mb for both species) and completeness (as determined by conserved BUSCO gene analysis). We were also able to resolve maternal and paternal haplotypes for 1/3 of the genome in both cases. By sequencing and assembling material from a single diploid individual, only two haplotypes are present, simplifying the assembly process compared to samples from multiple pooled individuals. This new low-input approach puts PacBio-based assemblies in reach for small, highly heterozygous organisms that comprise much of the diversity of life. The method presented here can be applied to samples with starting DNA amounts around 150 ng per 250 Mb – 600 Mb genome size.

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June 1, 2021

Streamlines SMRTbell library generation using addition-only, single tube strategy for all library types reduces time to results

We have streamlined the SMRTbell library generation protocols with improved workflows to deliver seamless end-to-end solutions from sample to analysis. A key improvement is the development of a single-tube reaction strategy that shortened hands-on time needed to generate each SMRTbell library, reduced time-consuming AM Pure purification steps, and minimized sample-handling induced gDNA damage to improve the integrity of long-insert SMRTbell templates for sequencing. The improved protocols support all large-insert genomic libraries, multiplexed microbial genomes, and amplicon sequencing. These advances enable completion of library preparation in less than a day (approximately 4 hours) and opens opportunities for automated library preparation for large-scale projects. Here we share data summarizing performance of the new SMRTbell Express Template Kit 2.0 representing our solutions for 10 kb and >50 kb large-insert genomic libraries, complete microbial genome assemblies, and high-throughput amplicon sequencing. The improved throughput of the Sequel System with read lengths up to 30 kb and high consensus accuracy (> 99.999% accuracy) makes sequencing with high-quality results increasingly assessible to the community.

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June 1, 2021

A low DNA input protocol for high-quality PacBio de novo genome assemblies

A high-quality reference genome is an essential tool for studying the genetics of traits and disease, organismal, comparative and conservation biology, and population genomics. PacBio Single Molecule, Real-Time (SMRT) Sequencing generates long reads with uniform coverage and high consensus accuracy, making it a powerful technology for de novo genome assembly. Improvements in throughput and concomitant reductions in cost have made PacBio an attractive core technology for many large genome initiatives. However, relatively high DNA input requirements (3 µg for standard library protocol) have placed PacBio out of reach for many projects on small organisms that may have lower DNA content or on projects with limited input DNA for other reasons. Here we present a modified SMRTbell library construction protocol without DNA shearing or size selection that can be used to generate a SMRTbell library from just 150 ng of starting genomic DNA. Remarkably, the protocol enables high quality de novo assemblies from single invertebrate individuals and is applied to taxonomically diverse samples. By sequencing and assembling material from a single diploid individual, only two haplotypes are present, simplifying the assembly process compared to samples from multiple pooled individuals. The libraries were run on the Sequel System with chemistry v3.0 and software v6.0, generating ~11 Gb of sequence per SMRT Cell with 10 hour movies, and followed by de novo genome assembly with FALCON. The resulting assemblies had high contiguity (contig N50s over 1 Mb) and completeness (as determined by conserved BUSCO gene analysis) when at least 30-fold unique molecular coverage is obtained. This new low-input approach now puts PacBio-based assemblies in reach for small highly heterozygous organisms that comprise much of the diversity of life. The method presented here is scalable and can be applied to samples with starting DNA amounts of 150 ng per 300 Mb genome size.

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June 1, 2021

Structural variant detection in crops using low-fold coverage long-read sequencing

Genomics studies have shown that the insertions, deletions, duplications, translocations, inversions, and tandem repeat expansions in the structural variant (SV) size range (>50 bp) contribute to the evolution of traits and often have significant associations with agronomically important phenotypes. However, most SVs are too small to detect with array comparative genomic hybridization and too large to reliably discover with short-read DNA sequencing. While de novo assembly is the most comprehensive way to identify variants in a genome, recent studies in human genomes show that PacBio SMRT Sequencing sensitively detects structural variants at low coverage. Here we present SV characterization in the major crop species Oryza sativa subsp. indica (rice) with low-fold coverage of long reads. In addition, we provide recommendations for sequencing and analysis for the application of this workflow to other important agricultural species.

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June 1, 2021

Every species can be a model: Reference-quality PacBio genomes from single insects

A high-quality reference genome is an essential resource for primary and applied research across the tree of life. Genome projects for small-bodied, non-model organisms such as insects face several unique challenges including limited DNA input quantities, high heterozygosity, and difficulty of culturing or inbreeding in the lab. Recent progress in PacBio library preparation protocols, sequencing throughput, and read accuracy address these challenges. We present several case studies including the Red Admiral (Vanessa atalanta), Monarch Butterfly (Danaus plexippus), and Anopheles malaria mosquitoes that highlight the benefits of sequencing single individuals for de novo genome assembly projects, and the ease at which these projects can be conducted by individual research labs. Sampled individuals may originate from lab colonies of interest to the research community or be sourced from the wild to better capture natural variation in a focal population. Where genomic DNA quantities are limited, the PacBio Low DNA Input Protocol requires ~100 ng of input DNA. Low DNA input samples with 500 Mb genome size or less can be multiplexed on a single SMRT Cell 8M on the Sequel II System. For samples with more abundant DNA quantity, size-selected libraries may be constructed to maximize sequencing yield. Both low DNA input and size-selected libraries can be used to generate HiFi reads, whose quality is Q20 or above (1% error or less) and lengths range from 10 – 25 kb. With HiFi reads, de novo assembly computation is greatly simplified relative to long read methods due to smaller sequence file sizes and more rapid analysis, resulting in highly accurate, contiguous, complete, and haplotype-resolved assemblies.

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June 1, 2021

Beyond Contiguity: Evaluating the accuracy of de novo genome assemblies

HiFi reads (>99% accurate, 15-20 kb) from the PacBio Sequel II System consistently provide complete and contiguous genome assemblies. In addition to completeness and contiguity, accuracy is of critical importance, as assembly errors complicate downstream analysis, particularly by disrupting gene frames. Metrics used to assess assembly accuracy include: 1) in-frame gene count, 2) kmer consistency, and 3) concordance to a benchmark, where discordances are interpreted as assembly errors. Genome in a Bottle (GIAB) provides a benchmark for the human genome with estimated accuracy of 99.9999% (Q60). Concordance for human HiFi assemblies exceeds Q50, which provides excellent genomes for downstream analysis, but presents a challenge that any new benchmark must significantly exceed Q50 or the discordance will represent the error rate of the benchmark. To establish benchmarks for Oryza sativa and Drosophila melanogaster, we collected draft references, Illumina short reads, and PacBio HiFi reads. By species, the benchmark was defined as regions of normal coverage that are not within 5 bp of a small variant or 50 bp of a structural variant. For both species, the benchmark regions span around 60% of the genome and HiFi assemblies achieve Q50 accuracy, which is notably more accurate than assemblies with other technologies and meets typical standards for a finished, reference-grade assembly. Here we present a protocol to generate benchmarks for any sample that rival the GIAB benchmark in accuracy. These benchmarks allow the comparison and improvement of genome assemblies and highlight the superior accuracy of assemblies generated with PacBio HiFi reads.

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June 1, 2021

A complete solution for high-quality genome annotation using the PacBio Iso-Seq method

The PacBio Iso-Seq method produces high-quality, full-length transcripts of up to 10 kb and longer and has been used to annotate many important plant and animal genomes. We describe here the full Iso-Seq ecosystem that enables researchers to achieve high-quality genome annotations. The Iso-Seq Express workflow is a 1-day protocol that requires only 60-300 ng of total RNA and supports multiplexing of different tissues. Sequencing on a single SMRT Cell 8M on the Sequel II System produces up to 4 million full-length reads, sufficient to exhaustively characterize a whole transcriptome on the order of 15,000-17,000 genes with 100,000 or more transcripts. Most importantly, the method is supported by a maturing suite of official and community-developed tools. The SMRT Link Iso-Seq application outputs high-quality (>99% accurate), full-length transcript sequences that can optionally be mapped to a reference genome for a single SMRT Cell worth of data in 6-9 hours. For example, the SQANTI2 tool classifies Iso-Seq transcripts against a reference annotation, filters potential library artifacts, and processes information from both long read-only and short read-based quantification. IsoPhase is a tool for identifying allele-specific isoform expression. Cogent has been used to process Iso-Seq transcripts in a genome-independent manner to assess genome assemblies. Finally, IsoAnnot is an up-and-coming tool for identifying differential isoform expression across different samples. We describe how these tools complement each other and provide guidelines to make the best use out of Iso-Seq data for understanding transcriptomes.

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June 1, 2021

A high-quality PacBio insect genome from 5 ng of input DNA

High-quality insect genomes are essential resources to understand insect biology and to combat them as disease vectors and agricultural pests. It is desirable to sequence a single individual for a reference genome to avoid complications from multiple alleles during de novo assembly. However, the small body size of many insects poses a challenge for the use of long-read sequencing technologies which often have high DNA-input requirements. The previously described PacBio Low DNA Input Protocol starts with ~100 ng of DNA and allows for high-quality assemblies of single mosquitoes among others and represents a significant step in reducing such requirements. Here, we describe a new library protocol with a further 20-fold reduction in the DNA input quantity. Starting with just 5 ng of high molecular weight DNA, we describe the successful sequencing and de novo genome assembly of a single male sandfly (Phlebotomus papatasi, the main vector of the Old World cutaneous leishmaniasis), using HiFi data generated on the PacBio Sequel II System and assembled with FALCON. The assembly shows a high degree of completeness (>97% of BUSCO genes are complete), contiguity (contig N50 of 1 Mb), and sequence accuracy (>98% of BUSCO genes without frameshift errors). This workflow has general utility for small-bodied insects and other plant and animal species for both focused research studies or in conjunction with large-scale genome projects.

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June 1, 2021

New advances in SMRT Sequencing facilitate multiplexing for de novo and structural variant studies

The latest advancements in Sequel II SMRT Sequencing have increased average read lengths up to 50% compared to Sequel II chemistry 1.0 which allows multiplexing of 2-3 small organisms (<500 Mb) such as insects and worms for producing reference quality assemblies, calling structural variants for up to 2 samples with ~3 Gb genomes, analysis of 48 microbial genomes, and up to 8 communities for metagenomic profiling in a single SMRT Cell 8M. With the improved processivity of the new Sequel II sequencing polymerase, more SMRTbell molecules reach rolling circle mode resulting in longer overall read lengths, thus allowing efficient detection of barcodes (up to 80%) in the SMRTbell templates. Multiplexing of genomes larger than microbial organisms is now achievable. In collaboration with the Wellcome Sanger Institute, we have developed a workflow for multiplexing two individual Anopheles coluzzii using as low as 150 ng genomic DNA per individual. The resulting assemblies had high contiguity (contig N50s over 3 Mb) and completeness (>98% of conserved genes) for both individuals. For microbial multiplexing, we multiplexed 48 microbes with varying complexities and sizes ranging 1.6-8.0 Mb in single SMRT Cell 8M. Using a new end-to-end analysis (Microbial Assembly Analysis, SMRT Link 8.0), assemblies resulted in complete circularized genomes (>200-fold coverage) and efficient detection of >3-200 kb plasmids. Finally, the long read lengths (>90 kb) allows detection of barcodes in large insert SMRTbell templates (>15 kb) thus facilitating multiplex of two human samples in 1 SMRT Cell 8M for detecting SVs, Indels and CNVs. Here, we present results and describe workflows for multiplexing samples for specific applications for SMRT Sequencing.

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