A new method for long-read sequencing of animal mitochondrial genomes: application to the identification of equine mitochondrial DNA variants

AbstractBackgroundWe present here an approach to sequence whole mitochondrial genomes using nanopore long-read sequencing. Our method relies on the selective elimination of nuclear DNA using an exonuclease treatment and on the amplification of circular mitochondrial DNA using a multiple displacement amplification step.ResultsWe optimized each preparative step to obtain a 100 million-fold enrichment of horse mitochondrial DNA relative to nuclear DNA. We sequenced these amplified mitochondrial DNA using nanopore sequencing technology and obtained mitochondrial DNA reads that represented up to half of the sequencing output. The sequence reads were 2.3 kb of mean length and provided an even coverage of the mitochondrial genome. Long-reads spanning half or more of the whole mtDNA provided a coverage that varied between 118X and 488X. Finally, we identified SNPs with a precision of 98.1%; recall of 85.2% and a F1-score of 0.912.ConclusionsOur analyses show that our method to amplify mtDNA and to sequence it using the nanopore technology is usable for mitochondrial DNA variant analysis. With minor modifications, this approach could easily be applied to other large circular DNA molecules.

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A new method for long-read sequencing of animal mitochondrial genomes: application to the identification of equine mitochondrial DNA variants

BMC Genomics ◽

10.1186/s12864-020-07183-9 ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

Sophie Dhorne-Pollet ◽

Eric Barrey ◽

Nicolas Pollet

Keyword(s):

Mitochondrial Dna ◽

Nuclear Dna ◽

Multiple Displacement Amplification ◽

Variant Calling ◽

Ground Truth ◽

Mitochondrial Genomes ◽

Long Reads ◽

Long Read ◽

Variant Analysis ◽

Mitochondrial Dna Variants

Abstract Background Mitochondrial DNA is remarkably polymorphic. This is why animal geneticists survey mitochondrial genomes variations for fundamental and applied purposes. We present here an approach to sequence whole mitochondrial genomes using nanopore long-read sequencing. Our method relies on the selective elimination of nuclear DNA using an exonuclease treatment and on the amplification of circular mitochondrial DNA using a multiple displacement amplification step. Results We optimized each preparative step to obtain a 100 million-fold enrichment of horse mitochondrial DNA relative to nuclear DNA. We sequenced these amplified mitochondrial DNA using nanopore sequencing technology and obtained mitochondrial DNA reads that represented up to half of the sequencing output. The sequence reads were 2.3 kb of mean length and provided an even coverage of the mitochondrial genome. Long-reads spanning half or more of the whole mtDNA provided a coverage that varied between 118X and 488X. We evaluated SNPs identified using these long-reads by Sanger sequencing as ground truth and found a precision of 100.0%; a recall of 93.1% and a F1-score of 0.964 using the Twilight horse mtDNA reference. The choice of the mtDNA reference impacted variant calling efficiency with F1-scores varying between 0.947 and 0.964. Conclusions Our method to amplify mtDNA and to sequence it using the nanopore technology is usable for mitochondrial DNA variant analysis. With minor modifications, this approach could easily be applied to other large circular DNA molecules.

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QAlign: Aligning nanopore reads accurately using current-level modeling

10.1101/862813 ◽

2019 ◽

Author(s):

Dhaivat Joshi ◽

Shunfu Mao ◽

Sreeram Kannan ◽

Suhas Diggavi

Keyword(s):

Reference Genome ◽

Genomic Analysis ◽

Vital Role ◽

High Error Rate ◽

Sequencing Technology ◽

Long Reads ◽

A Genome ◽

Long Read ◽

Nanopore Sequencer ◽

Sequencing Process

AbstractMotivationEfficient and accurate alignment of DNA / RNA sequence reads to each other or to a reference genome / transcriptome is an important problem in genomic analysis. Nanopore sequencing has emerged as a major sequencing technology and many long-read aligners have been designed for aligning nanopore reads. However, the high error rate makes accurate and efficient alignment difficult. Utilizing the noise and error characteristics inherent in the sequencing process properly can play a vital role in constructing a robust aligner. In this paper, we design QAlign, a pre-processor that can be used with any long-read aligner for aligning long reads to a genome / transcriptome or to other long reads. The key idea in QAlign is to convert the nucleotide reads into discretized current levels that capture the error modes of the nanopore sequencer before running it through a sequence aligner.ResultsWe show that QAlign is able to improve alignment rates from around 80% up to 90% with nanopore reads when aligning to the genome. We also show that QAlign improves the average overlap quality by 9.2%, 2.5% and 10.8% in three real datasets for read-to-read alignment. Read-to-transcriptome alignment rates are improved from 51.6% to 75.4% and 82.6% to 90% in two real datasets.Availabilityhttps://github.com/joshidhaivat/QAlign.git

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Impact of Chimera-less Long Reads on Metagenomics of Human Gut Viromes Treated With Multiple Displacement Amplification

10.21203/rs.3.rs-58640/v1 ◽

2020 ◽

Author(s):

Yuya Kiguchi ◽

Suguru Nishijima ◽

Naveen Kumar ◽

Masahira Hattori ◽

Wataru Suda

Keyword(s):

De Novo ◽

Multiple Displacement Amplification ◽

Read Length ◽

Human Gut ◽

Average Read Length ◽

Sequencing Technologies ◽

Genomic Complexity ◽

Long Reads ◽

Long Read ◽

The Impact

Abstract Background: The ecological and biological features of the indigenous phage community (virome) in the human gut microbiome are poorly understood, possibly due to many fragmented contigs and fewer complete genomes based on conventional short-read metagenomics. Long-read sequencing technologies have attracted attention as an alternative approach to reconstruct long and accurate contigs from microbial communities. However, the impact of long-read metagenomics on human gut virome analysis has not been well evaluated. Results: Here we present chimera-less PacBio long-read metagenomics of multiple displacement amplification (MDA)-treated human gut virome DNA. The method included the development of a novel bioinformatics tool, SACRA (Split Amplified Chimeric Read Algorithm), which efficiently detects and splits numerous chimeric reads in PacBio reads from the MDA-treated virome samples. SACRA treatment of PacBio reads from five samples markedly reduced the average chimera ratio from 72 to 1.5%, generating chimera-less PacBio reads with an average read-length of 1.8 kb. De novo assembly of the chimera-less long reads generated contigs with an average N50 length of 11.1 kb, whereas those of MiSeq short reads from the same samples were 0.7 kb, dramatically improving contig extension. Alignment of both contig sets generated 378 high-quality merged contigs (MCs) composed of the minimum scaffolds of 434 MiSeq and 637 PacBio contigs, respectively, and also identified numerous MiSeq short fragmented contigs ≤500 bp additionally aligned to MCs, which possibly originated from a small fraction of MiSeq chimeric reads. The alignment also revealed that fragmentations of the scaffolded MiSeq contigs were caused primarily by genomic complexity of the community, including local repeats, hypervariable regions, and highly conserved sequences in and between the phage genomes. We identified 142 complete and near-complete phage genomes including 108 novel genomes, varying from 5 to 185 kb in length, the majority of which were predicted to be Microviridae phages including several variants with homologous but distinct genomes, which were fragmented in MiSeq contigs. Conclusions: Long-read metagenomics coupled with SACRA provides an improved method to reconstruct accurate and extended phage genomes from MDA-treated virome samples of the human gut, and potentially from other environmental virome samples.

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Long-read assemblies reveal structural diversity in genomes of organelles - an example with Acacia pycnantha

10.1101/2020.12.22.423164 ◽

2020 ◽

Author(s):

Anna E. Syme ◽

Todd G.B. McLay ◽

Frank Udovicic ◽

David J. Cantrill ◽

Daniel J. Murphy

Keyword(s):

Mitochondrial Genome ◽

Chloroplast Genome ◽

De Novo ◽

Genomic Structure ◽

Structural Diversity ◽

Mitochondrial Genomes ◽

Long Reads ◽

Organelle Genomes ◽

Long Read ◽

Assembly Algorithms

AbstractAlthough organelle genomes are typically represented as single, static, circular molecules, there is evidence that the chloroplast genome exists in two structural haplotypes and that the mitochondrial genome can display multiple circular, linear or branching forms. We sequenced and assembled chloroplast and mitochondrial genomes of the Golden Wattle, Acacia pycnantha, using long reads, iterative baiting to extract organelle-only reads, and several assembly algorithms to explore genomic structure. Using a de novo assembly approach agnostic to previous hypotheses about structure, we found different assemblies revealed contrasting arrangements of genomic segments; a hypothesis supported by mapped reads spanning alternate paths.

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Long-reads are revolutionizing 20 years of insect genome sequencing

10.1101/2021.02.14.431146 ◽

2021 ◽

Author(s):

Scott Hotaling ◽

John S. Sproul ◽

Jacqueline Heckenhauer ◽

Ashlyn Powell ◽

Amanda M. Larracuente ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Nuclear Genome ◽

Sequencing Technology ◽

Assembly Quality ◽

Insect Genome ◽

Long Reads ◽

Long Read ◽

Field Perspective ◽

Genome Assemblies ◽

The Impact

The first insect genome (Drosophila melanogaster) was published two decades ago. Today, nuclear genome assemblies are available for a staggering 601 different insects representing 20 orders. Here, we analyzed the best assembly for each insect and provide a “state of the field” perspective, emphasizing taxonomic representation, assembly quality, gene completeness, and sequencing technology. We show that while genomic efforts have been biased towards specific groups (e.g., Diptera), assemblies are generally contiguous with gene regions intact. Most notable, however, has been the impact of long-read sequencing; assemblies that incorporate long-reads are ∼48x more contiguous than those that do not.

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Long-Read Sequencing of the Zebrafish Genome Reorganizes Genomic Architecture

10.1101/2021.08.27.457855 ◽

2021 ◽

Author(s):

Yelena Chernyavskaya ◽

Xiaofei Zhang ◽

Jinze Liu ◽

Jessica S. Blackburn

Keyword(s):

Low Complexity ◽

Zebrafish Genome ◽

Nanopore Sequencing ◽

Sequencing Technology ◽

Short Read ◽

Short Read Sequencing ◽

Genomic Landscape ◽

Long Reads ◽

Long Read ◽

Sequencing Platforms

Nanopore sequencing technology has revolutionized the field of genome biology with its ability to generate extra-long reads that can resolve regions of the genome that were previously inaccessible to short-read sequencing platforms. Although long-read sequencing has been used to resolve several vertebrate genomes, a nanopore-based zebrafish assembly has not yet been released. Over 50% of the zebrafish genome consists of difficult to map, highly repetitive, low complexity elements that pose inherent problems for short-read sequencers and assemblers. We used nanopore sequencing to improve upon and resolve the issues plaguing the current zebrafish reference assembly (GRCz11). Our long-read assembly improved the current resolution of the reference genome by identifying 1,697 novel insertions and deletions over 1Kb in length and placing 106 previously unlocalized scaffolds. We also discovered additional sites of retrotransposon integration previously unreported in GRCz11 and observed their expression in adult zebrafish under physiologic conditions, implying they have active mobility in the zebrafish genome and contribute to the ever-changing genomic landscape.

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QAlign: aligning nanopore reads accurately using current-level modeling

Bioinformatics ◽

10.1093/bioinformatics/btaa875 ◽

2020 ◽

Author(s):

Dhaivat Joshi ◽

Shunfu Mao ◽

Sreeram Kannan ◽

Suhas Diggavi

Keyword(s):

Reference Genome ◽

Genomic Analysis ◽

Vital Role ◽

Supplementary Information ◽

Sequencing Technology ◽

Long Reads ◽

A Genome ◽

Long Read ◽

Nanopore Sequencer ◽

Sequencing Process

Abstract Motivation Efficient and accurate alignment of DNA/RNA sequence reads to each other or to a reference genome/transcriptome is an important problem in genomic analysis. Nanopore sequencing has emerged as a major sequencing technology and many long-read aligners have been designed for aligning nanopore reads. However, the high error rate makes accurate and efficient alignment difficult. Utilizing the noise and error characteristics inherent in the sequencing process properly can play a vital role in constructing a robust aligner. In this article, we design QAlign, a pre-processor that can be used with any long-read aligner for aligning long reads to a genome/transcriptome or to other long reads. The key idea in QAlign is to convert the nucleotide reads into discretized current levels that capture the error modes of the nanopore sequencer before running it through a sequence aligner. Results We show that QAlign is able to improve alignment rates from around 80% up to 90% with nanopore reads when aligning to the genome. We also show that QAlign improves the average overlap quality by 9.2, 2.5 and 10.8% in three real datasets for read-to-read alignment. Read-to-transcriptome alignment rates are improved from 51.6% to 75.4% and 82.6% to 90% in two real datasets. Availability and implementation https://github.com/joshidhaivat/QAlign.git. Supplementary information Supplementary data are available at Bioinformatics online.

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Transcriptome assembly from long-read RNA-seq alignments with StringTie2

10.1101/694554 ◽

2019 ◽

Author(s):

Sam Kovaka ◽

Aleksey V. Zimin ◽

Geo M. Pertea ◽

Roham Razaghi ◽

Steven L. Salzberg ◽

...

Keyword(s):

Single Molecule ◽

Transcriptome Assembly ◽

Rna Seq ◽

High Error Rate ◽

Sequencing Technology ◽

Ability To Work ◽

Single Molecule Sequencing ◽

Long Reads ◽

Long Read

AbstractRNA sequencing using the latest single-molecule sequencing instruments produces reads that are thousands of nucleotides long. The ability to assemble these long reads can greatly improve the sensitivity of long-read analyses. Here we present StringTie2, a reference-guided transcriptome assembler that works with both short and long reads. StringTie2 includes new computational methods to handle the high error rate of long-read sequencing technology, which previous assemblers could not tolerate. It also offers the ability to work with full-length super-reads assembled from short reads, which further improves the quality of assemblies. On 33 short-read datasets from humans and two plant species, StringTie2 is 47.3% more precise and 3.9% more sensitive than Scallop. On multiple long read datasets, StringTie2 on average correctly assembles 8.3 and 2.6 times as many transcripts as FLAIR and Traphlor, respectively, with substantially higher precision. StringTie2 is also faster and has a smaller memory footprint than all comparable tools.

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The alternative reality of plant mitochondrial DNA

10.1101/564278 ◽

2019 ◽

Cited By ~ 5

Author(s):

Alexander Kozik ◽

Beth A. Rowan ◽

Dean Lavelle ◽

Lidija Berke ◽

M. Eric Schranz ◽

...

Keyword(s):

Mitochondrial Dna ◽

Genome Structure ◽

Structural Diversity ◽

Data Availability ◽

Mitochondrial Genomes ◽

Physical Analysis ◽

Sequencing Data ◽

True Nature ◽

Plant Mitochondrial Dna ◽

Long Read

ABSTRACTPlant mitochondrial genomes are usually assembled and displayed as circular maps based on the widely-held assumption that circular genome molecules are the primary form of mitochondrial DNA, despite evidence to the contrary. Many plant mitochondrial genomes have one or more pairs of large repeats that can act as sites for inter- or intramolecular recombination, leading to multiple alternative genomic arrangements (isoforms). Most mitochondrial genomes have been assembled using methods that were unable to capture the complete spectrum of isoforms within a species, leading to an incomplete inference of their structure and recombinational activity. To document and investigate underlying reasons for structural diversity in plant mitochondrial DNA, we used long-read (PacBio) and short-read (Illumina) sequencing data to assemble and compare mitochondrial genomes of domesticated (Lactuca sativa) and wild (L. saligna and L. serriola) lettuce species. This allowed us to characterize a comprehensive, complex set of isoforms within each species and to compare genome structures between species. Physical analysis of L. sativa mtDNA molecules by fluorescence microscopy revealed a variety of linear, branched linear, and circular structures. The mitochondrial genomes for L. sativa and L. serriola were identical in sequence and arrangement, and differed substantially from L. saligna, indicating that the mitochondrial genome structure did not change during domestication. From the isoforms evident in our data, we inferred that recombination occurs at repeats of all sizes at variable frequencies. The differences in genome structure between L. saligna and the two other lettuce species can be largely explained by rare recombination events that rearrange the structure. Our data demonstrate that representations of plant mitochondrial DNA as simple, genome-sized circular molecules are not accurate descriptions of their true nature and that in reality plant mitochondrial DNA is a complex, dynamic mixture of forms.Data AvailabilityBioProject: Organellar genomes of cultivated and wild lettuce (Lactuca) varieties PRJNA508811 https://www.ncbi.nlm.nih.gov/bioproject/508811 and other accessions as indicated through the text and supplemental data.FundingNSF grant MCB-1413152 to ACC and support from UC Davis to RWM.

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