A technology-agnostic long-read analysis pipeline for transcriptome discovery and quantification

Mapping Intimacies ◽

10.1101/672931 ◽

2019 ◽

Cited By ~ 15

Author(s):

Dana Wyman ◽

Gabriela Balderrama-Gutierrez ◽

Fairlie Reese ◽

Shan Jiang ◽

Sorena Rahmanian ◽

...

Keyword(s):

Cost Effective ◽

Short Read ◽

Disease States ◽

Novel Transcript ◽

Gm12878 Cell ◽

Long Read ◽

Transcript Reconstruction ◽

Uniform Manner ◽

Full Length Transcript ◽

Sequencing Platforms

ABSTRACTAlternative splicing is widely acknowledged to be a crucial regulator of gene expression and is a key contributor to both normal developmental processes and disease states. While cost-effective and accurate for quantification, short-read RNA-seq lacks the ability to resolve full-length transcript isoforms despite increasingly sophisticated computational methods. Long-read sequencing platforms such as Pacific Biosciences (PacBio) and Oxford Nanopore (ONT) bypass the transcript reconstruction challenges of short reads. Here we introduce TALON, the ENCODE4 pipeline for platform-independent analysis of long-read transcriptomes. We apply TALON to the GM12878 cell line and show that while both PacBio and ONT technologies perform well at full-transcript discovery and quantification, each displayed distinct technical artifacts. We further apply TALON to mouse hippocampus and cortex transcriptomes and find that 422 genes found in these regions have more reads associated with novel isoforms than with annotated ones. We demonstrate that TALON is a capable of tracking both known and novel transcript models as well as their expression levels across datasets for both simple studies and in larger projects. These properties will enable TALON users to move beyond the limitations of short-read data to perform isoform discovery and quantification in a uniform manner on existing and future long-read platforms.

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Ultra-low input single tube linked-read library method enables short-read NGS systems to generate highly accurate and economical long-range sequencing information for de novo genome assembly and haplotype phasing

10.1101/852947 ◽

2019 ◽

Cited By ~ 3

Author(s):

Zhoutao Chen ◽

Long Pham ◽

Tsai-Chin Wu ◽

Guoya Mo ◽

Yu Xia ◽

...

Keyword(s):

Long Range ◽

De Novo ◽

Low Cost ◽

Cost Effective ◽

De Novo Genome Assembly ◽

Short Read ◽

Single Tube ◽

Haplotype Phasing ◽

A Genome ◽

Long Read

AbstractLong-range sequencing information is required for haplotype phasing, de novo assembly and structural variation detection. Current long-read sequencing technologies can provide valuable long-range information but at a high cost with low accuracy and high DNA input requirement. We have developed a single-tube Transposase Enzyme Linked Long-read Sequencing (TELL-Seq™) technology, which enables a low-cost, high-accuracy and high-throughput short-read next generation sequencer to routinely generate over 100 Kb long-range sequencing information with as little as 0.1 ng input material. In a PCR tube, millions of clonally barcoded beads are used to uniquely barcode long DNA molecules in an open bulk reaction without dilution and compartmentation. The barcode linked reads are used to successfully assemble genomes ranging from microbes to human. These linked-reads also generate mega-base-long phased blocks and provide a cost-effective tool for detecting structural variants in a genome, which are important to identify compound heterozygosity in recessive Mendelian diseases and discover genetic drivers and diagnostic biomarkers in cancers.

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CaBagE: A Cas9-based Background Elimination strategy for targeted, long-read DNA sequencing

PLoS ONE ◽

10.1371/journal.pone.0241253 ◽

2021 ◽

Vol 16 (4) ◽

pp. e0241253

Author(s):

Amelia D. Wallace ◽

Thomas A. Sasani ◽

Jordan Swanier ◽

Brooke L. Gates ◽

Jeff Greenland ◽

...

Keyword(s):

Dna Sequencing ◽

Tandem Repeats ◽

Short Read ◽

Background Elimination ◽

Sequencing Technologies ◽

Long Reads ◽

Long Read ◽

Repeat Expansions ◽

Sequencing Platforms ◽

Als Patients

A substantial fraction of the human genome is difficult to interrogate with short-read DNA sequencing technologies due to paralogy, complex haplotype structures, or tandem repeats. Long-read sequencing technologies, such as Oxford Nanopore’s MinION, enable direct measurement of complex loci without introducing many of the biases inherent to short-read methods, though they suffer from relatively lower throughput. This limitation has motivated recent efforts to develop amplification-free strategies to target and enrich loci of interest for subsequent sequencing with long reads. Here, we present CaBagE, a method for target enrichment that is efficient and useful for sequencing large, structurally complex targets. The CaBagE method leverages the stable binding of Cas9 to its DNA target to protect desired fragments from digestion with exonuclease. Enriched DNA fragments are then sequenced with Oxford Nanopore’s MinION long-read sequencing technology. Enrichment with CaBagE resulted in a median of 116X coverage (range 39–416) of target loci when tested on five genomic targets ranging from 4-20kb in length using healthy donor DNA. Four cancer gene targets were enriched in a single reaction and multiplexed on a single MinION flow cell. We further demonstrate the utility of CaBagE in two ALS patients with C9orf72 short tandem repeat expansions to produce genotype estimates commensurate with genotypes derived from repeat-primed PCR for each individual. With CaBagE there is a physical enrichment of on-target DNA in a given sample prior to sequencing. This feature allows adaptability across sequencing platforms and potential use as an enrichment strategy for applications beyond sequencing. CaBagE is a rapid enrichment method that can illuminate regions of the ‘hidden genome’ underlying human disease.

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High-throughput annotation of full-length long noncoding RNAs with Capture Long-Read Sequencing

10.1101/105064 ◽

2017 ◽

Cited By ~ 4

Author(s):

Julien Lagarde ◽

Barbara Uszczynska-Ratajczak ◽

Silvia Carbonell ◽

SÍlvia Pérez-Lluch ◽

Amaya Abad ◽

...

Keyword(s):

High Throughput ◽

Single Molecule ◽

Noncoding Rnas ◽

Splice Junction ◽

Long Noncoding Rnas ◽

Full Length ◽

Novel Transcript ◽

Mouse Tissues ◽

Long Read ◽

Full Length Transcript

AbstractAccurate annotations of genes and their transcripts is a foundation of genomics, but no annotation technique presently combines throughput and accuracy. As a result, reference gene collections remain incomplete: many gene models are fragmentary, while thousands more remain uncatalogued–particularly for long noncoding RNAs (lncRNAs). To accelerate lncRNA annotation, the GENCODE consortium has developed RNA Capture Long Seq (CLS), combining targeted RNA capture with third-generation long-read sequencing. We present an experimental re-annotation of the GENCODE intergenic lncRNA population in matched human and mouse tissues, resulting in novel transcript models for 3574 / 561 gene loci, respectively. CLS approximately doubles the annotated complexity of targeted loci, outperforming existing short-read techniques. Full-length transcript models produced by CLS enable us to definitively characterize the genomic features of lncRNAs, including promoter- and gene-structure, and protein-coding potential. Thus CLS removes a longstanding bottleneck of transcriptome annotation, generating manual-quality full-length transcript models at high-throughput scales.Abbreviationsbpbase pairFLfull lengthntnucleotideROIread of insert, i.e. PacBio readSJsplice junctionSMRTsingle-molecule real-timeTMtranscript model

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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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Complete and high-quality genomes of novel microbial species from a meromictic lake using a workflow combining long- and short-read sequencing platforms

10.1101/2021.05.07.443067 ◽

2021 ◽

Author(s):

Yu-Hsiang Chen ◽

Pei-Wen Chiang ◽

Denis Yu Rogozin ◽

Andrey Georgievich Degermendzhy ◽

Hsiu-Hui Chiu ◽

...

Keyword(s):

Novel Species ◽

Bacterial Genome ◽

Meromictic Lake ◽

High Quality ◽

Good Opportunity ◽

Short Read ◽

Sequencing Technologies ◽

Complete Genomes ◽

Long Read ◽

Sequencing Platforms

Background: Most of Earth's bacteria have yet to be cultivated. The metabolic and functional potentials of these uncultivated microorganisms thus remain mysterious, and the metagenome-assembled genome (MAG) approach is the most robust method for uncovering these potentials. However, MAGs discovered by conventional metagenomic assembly and binning methods are usually highly fragmented genomes with heterogeneous sequence contamination, and this affects the accuracy and sensitivity of genomic analyses. Though the maturation of long-read sequencing technologies provides a good opportunity to fix the problem of highly fragmented MAGs as mentioned above, the method's error-prone nature causes severe problems of long-read-alone metagenomics. Hence, methods are urgently needed to retrieve MAGs by a combination of both long- and short-read technologies to advance genome-centric metagenomics. Results: In this study, we combined Illumina and Nanopore data to develop a new workflow to reconstruct 233 MAGs-six novel bacterial orders, 20 families, 66 genera, and 154 species-from Lake Shunet, a secluded meromictic lake in Siberia. Those new MAGs were underrepresented or undetectable in other MAGs studies using metagenomes from human or other common organisms or habitats. Using this newly developed workflow and strategy, the average N50 of reconstructed MAGs greatly increased 10-40-fold compared to when the conventional Illumina assembly and binning method were used. More importantly, six complete MAGs were recovered from our datasets, five of which belong to novel species. We used these as examples to demonstrate many novel and intriguing genomic characteristics discovered in these newly complete genomes and proved the importance of high-quality complete MAGs in microbial genomics and metagenomics studies. Conclusions: The results show that it is feasible to apply our workflow with a few additional long reads to recover numerous complete and high-quality MAGs from short-read metagenomes of high microbial diversity environment samples. The unique features we identified from five complete genomes highlight the robustness of this method in genome-centric metagenomic research. The recovery of 154 novel species MAGs from a rarely explored lake greatly expands the current bacterial genome encyclopedia and broadens our knowledge by adding new genomic characteristics of bacteria. It demonstrates a strong need to recover MAGs from diverse unexplored habitats in the search for microbial dark matter.

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Long-TUC-seq is a robust method for quantification of metabolically labeled full-length isoforms

10.1101/2020.05.01.073296 ◽

2020 ◽

Author(s):

Sorena Rahmanian ◽

Gabriela Balderrama-Gutierrez ◽

Dana Wyman ◽

Cassandra Joan McGill ◽

Kim Nguyen ◽

...

Keyword(s):

Steady State ◽

Metabolic Labeling ◽

Rna Seq ◽

Robust Method ◽

Mrna Transcription ◽

Short Read ◽

Gm12878 Cell ◽

Gene Level ◽

Long Read ◽

Labeled Rna

ABSTRACTThe steady state expression of each gene is the result of a dynamic transcription and degradation of that gene. While regular RNA-seq methods only measure steady state expression levels, RNA-seq of metabolically labeled RNA identifies transcripts that were transcribed during the window of metabolic labeling. Whereas short-read RNA sequencing can identify metabolically labeled RNA at the gene level, long-read sequencing provides much better resolution of isoform-level transcription. Here we combine thiouridine-to-cytosine conversion (TUC) with PacBio long-read sequencing to study the dynamics of mRNA transcription in the GM12878 cell line. We show that using long-TUC-seq, we can detect metabolically labeled mRNA of distinct isoforms more reliably than using short reads. Long-TUC-seq holds the promise of capturing isoform dynamics robustly and without the need for enrichment.

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Comparison of long-read sequencing technologies in the hybrid assembly of complex bacterial genomes

10.1101/530824 ◽

2019 ◽

Cited By ~ 3

Author(s):

Nicola De Maio ◽

Liam P. Shaw ◽

Alasdair Hubbard ◽

Sophie George ◽

Nick Sanderson ◽

...

Keyword(s):

Bacterial Genome ◽

Hybrid Assembly ◽

Bacterial Genomes ◽

Short Read ◽

Short Reads ◽

Genome Reconstruction ◽

Long Reads ◽

Oxford Nanopore ◽

Long Read ◽

Sequencing Platforms

ABSTRACTIllumina sequencing allows rapid, cheap and accurate whole genome bacterial analyses, but short reads (<300 bp) do not usually enable complete genome assembly. Long read sequencing greatly assists with resolving complex bacterial genomes, particularly when combined with short-read Illumina data (hybrid assembly). However, it is not clear how different long-read sequencing methods impact on assembly accuracy. Relative automation of the assembly process is also crucial to facilitating high-throughput complete bacterial genome reconstruction, avoiding multiple bespoke filtering and data manipulation steps. In this study, we compared hybrid assemblies for 20 bacterial isolates, including two reference strains, using Illumina sequencing and long reads from either Oxford Nanopore Technologies (ONT) or from SMRT Pacific Biosciences (PacBio) sequencing platforms. We chose isolates from the Enterobacteriaceae family, as these frequently have highly plastic, repetitive genetic structures and complete genome reconstruction for these species is relevant for a precise understanding of the epidemiology of antimicrobial resistance. We de novo assembled genomes using the hybrid assembler Unicycler and compared different read processing strategies. Both strategies facilitate high-quality genome reconstruction. Combining ONT and Illumina reads fully resolved most genomes without additional manual steps, and at a lower consumables cost per isolate in our setting. Automated hybrid assembly is a powerful tool for complete and accurate bacterial genome assembly.IMPACT STATEMENTIllumina short-read sequencing is frequently used for tasks in bacterial genomics, such as assessing which species are present within samples, checking if specific genes of interest are present within individual isolates, and reconstructing the evolutionary relationships between strains. However, while short-read sequencing can reveal significant detail about the genomic content of bacterial isolates, it is often insufficient for assessing genomic structure: how different genes are arranged within genomes, and particularly which genes are on plasmids – potentially highly mobile components of the genome frequently carrying antimicrobial resistance elements. This is because Illumina short reads are typically too short to span repetitive structures in the genome, making it impossible to accurately reconstruct these repetitive regions. One solution is to complement Illumina short reads with long reads generated with SMRT Pacific Biosciences (PacBio) or Oxford Nanopore Technologies (ONT) sequencing platforms. Using this approach, called ‘hybrid assembly’, we show that we can automatically fully reconstruct complex bacterial genomes of Enterobacteriaceae isolates in the majority of cases (best-performing method: 17/20 isolates). In particular, by comparing different methods we find that using the assembler Unicycler with Illumina and ONT reads represents a low-cost, high-quality approach for reconstructing bacterial genomes using publicly available software.DATA SUMMARYRaw sequencing data and assemblies have been deposited in NCBI under BioProject Accession PRJNA422511 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA422511). We confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

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High-throughput targeted long-read single cell sequencing reveals the clonal and transcriptional landscape of lymphocytes

10.1101/424945 ◽

2018 ◽

Cited By ~ 1

Author(s):

Mandeep Singh ◽

Ghamdan Al-Eryani ◽

Shaun Carswell ◽

James M. Ferguson ◽

James Blackburn ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

High Throughput ◽

Clonal Evolution ◽

Primary Tumour ◽

Transcriptome Profiling ◽

Cost Effective ◽

Short Read ◽

Wide Range ◽

Long Read

AbstractHigh-throughput single-cell RNA-Sequencing is a powerful technique for gene expression profiling of complex and heterogeneous cellular populations such as the immune system. However, these methods only provide short-read sequence from one end of a cDNA template, making them poorly suited to the investigation of gene-regulatory events such as mRNA splicing, adaptive immune responses or somatic genome evolution. To address this challenge, we have developed a method that combines targeted long-read sequencing with short-read based transcriptome profiling of barcoded single cell libraries generated by droplet-based partitioning. We use Repertoire And Gene Expression sequencing (RAGE-seq) to accurately characterize full-length T cell (TCR) and B cell (BCR) receptor sequences and transcriptional profiles of more than 7,138 lymphocytes sampled from the primary tumour and draining lymph node of a breast cancer patient. With this method we show that somatic mutation, alternate splicing and clonal evolution of T and B lymphocytes can be tracked across these tissue compartments. Our results demonstrate that RAGE-Seq is an accessible and cost-effective method for high-throughput deep single cell profiling, applicable to a wide range of biological challenges.

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HIGH RESOLUTION ANNOTATION OF ZEBRAFISH TRANSCRIPTOME USING LONG-READ SEQUENCING

10.1101/174821 ◽

2017 ◽

Cited By ~ 1

Author(s):

German Nudelman ◽

Antonio Frasca ◽

Brandon Kent ◽

Kirsten Edepli-Sadler ◽

Stuart C. Sealfon ◽

...

Keyword(s):

Model Organism ◽

Sequencing Data ◽

High Confidence ◽

Short Read ◽

Short Read Sequencing ◽

Functional Conservation ◽

Transcript Quantification ◽

Before And After ◽

Long Read ◽

Sequencing Platforms

ABSTRACTWith the emergence of zebrafish as an important model organism, a concerted effort has been made to study its transcriptome. This effort is limited, however, by gaps in zebrafish annotation, which are especially pronounced concerning transcripts dynamically expressed during zygotic genome activation (ZGA). To date, short read sequencing has been the principal technology for zebrafish transcriptome annotation. In part because these sequence reads are too short for assembly methods to resolve the full complexity of the transcriptome, the current annotation is rudimentary. By providing direct observation of full-length transcripts, recently refined long-read sequencing platforms can dramatically improve annotation coverage and accuracy. Here, we leveraged the SMRT platform to study transcriptome of zebrafish embryos before and after ZGA. Our analysis revealed additional novelty and complexity in the zebrafish transcriptome, identifying 2748 high confidence novel transcripts that originated from previously unannotated loci and 1835 high confidence new isoforms in previously annotated genes. We validated these findings using a suite of computational approaches including structural prediction, sequence homology and functional conservation analyses, as well as by confirmatory transcript quantification with short-read sequencing data. Our analyses provided insight into new homologs and paralogs of functionally important proteins and non-coding RNAs, isoform switching occurrences and different classes of novel splicing events. Several novel isoforms representing distinct splicing events were validated through PCR experiments, including the discovery and validation of a novel 8 kb transcript spanning multiple miR-430 elements, an important driver of early development. Our study provides a significantly improved zebrafish transcriptome annotation resource.

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Long-read viral metagenomics enables capture of abundant and microdiverse viral populations and their niche-defining genomic islands

10.1101/345041 ◽

2018 ◽

Cited By ~ 3

Author(s):

Joanna Warwick-Dugdale ◽

Natalie Solonenko ◽

Karen Moore ◽

Lauren Chittick ◽

Ann C. Gregory ◽

...

Keyword(s):

High Throughput ◽

Low Cost ◽

Cost Effective ◽

Host Community ◽

Genomic Islands ◽

Viral Metagenomics ◽

Viral Gene ◽

Bioinformatics Pipeline ◽

Short Read ◽

Long Read

AbstractMarine viruses impact global biogeochemical cycles via their influence on host community structure and function, yet our understanding of viral ecology is constrained by limitations in culturing of important hosts and the lack of a ‘universal’ gene to facilitate community surveys. Short-read viral metagenomic studies have provided clues to viral function and first estimates of global viral gene abundance and distribution. However, short-read assemblies are confounded by populations with high levels of strain evenness and nucleotide diversity (microdiversity), limiting assembly of some of the most abundant viruses on Earth. Assembly across genomic islands which likely contain niche-defining genes that drive ecological speciation is also challenging. While such populations and features are successfully captured by single-virus genomics and fosmid-based approaches, both techniques require considerable cost and technical expertise. Here we established a low-cost, low-input, high throughput alternative method for improving assembly of viral metagenomics using long read technology. Named ‘VirION’ (Viral, long-read metagenomics via MinION sequencing), our sequencing approach and complementary bioinformatics pipeline (i) increased number and completeness of assembled viral genomes compared to short-read sequencing methods; (ii) captured populations of abundant viruses with high microdiversity missed by short-read methods and (iii) captured more and longer genomic islands than short-read methods. Thus, VirION provides a high throughput and cost-effective alternative to fosmid and single-virus genomic approaches to more comprehensively explore viral communities in nature.

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