scholarly journals Nanopore native RNA sequencing of a human poly(A) transcriptome: RNA extraction, cDNA conversion and direct RNA and cDNA library preparation for Oxford Nanopore

2019 ◽  
Author(s):  
Rachael E. Workman ◽  
Alison D. Tang ◽  
Paul S. Tang ◽  
Miten Jain ◽  
John R. Tyson ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Rna Extraction ◽  
Human Cell Line ◽  
Cdna Libraries ◽  
Library Preparation ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Sequencing Strategy ◽  
Oxford Nanopore Technologies ◽  
Cdna Library Preparation

Abstract High throughput cDNA sequencing technologies have dramatically advanced our understanding of transcriptome complexity and regulation. However, these methods lose information contained in biological RNA because the copied reads are often short and because modifications are not carried forward in cDNA. We address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies (ONT). Our study focused on poly(A) RNA from the human cell line GM12878, generating 9.9 million aligned sequence reads. These native RNA reads had an aligned N50 length of 1294 bases, and a maximum aligned length of over 21,000 bases. A total of 78,199 high-confidence isoforms were identified by combining long nanopore reads with short higher accuracy Illumina reads. We describe methods for extracting intact RNA, poly-A selection, cDNA conversion for a portion of sample, and library preparation for both direct RNA and cDNA libraries.

scholarly journals Nanopore native RNA sequencing of a human poly(A) transcriptome

2018 ◽  
Author(s):  
Rachael E. Workman ◽  
Alison D. Tang ◽  
Paul S. Tang ◽  
Miten Jain ◽  
John R. Tyson ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Tail Length ◽  
Human Cell Line ◽  
Cdna Sequencing ◽  
High Confidence ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Rna Biology ◽  
Sequencing Strategy ◽  
Oxford Nanopore Technologies

ABSTRACTHigh throughput cDNA sequencing technologies have dramatically advanced our understanding of transcriptome complexity and regulation. However, these methods lose information contained in biological RNA because the copied reads are often short and because modifications are not carried forward in cDNA. We address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies (ONT). Our study focused on poly(A) RNA from the human cell line GM12878, generating 9.9 million aligned sequence reads. These native RNA reads had an aligned N50 length of 1294 bases, and a maximum aligned length of over 21,000 bases. A total of 78,199 high-confidence isoforms were identified by combining long nanopore reads with short higher accuracy Illumina reads. We describe strategies for assessing 3′ poly(A) tail length, base modifications and transcript haplotypes from nanopore RNA data. Together, these nanopore-based techniques are poised to deliver new insights into RNA biology.DISCLOSURESMA holds shares in Oxford Nanopore Technologies (ONT). MA is a paid consultant to ONT. REW, WT, TG, JRT, JQ, NJL, JTS, NS, AB, MA, HEO, MJ, and ML received reimbursement for travel, accommodation and conference fees to speak at events organised by ONT. NL has received an honorarium to speak at an ONT company meeting. WT has two patents (8,748,091 and 8,394,584) licensed to Oxford Nanopore. JTS, ML and MA received research funding from ONT.

scholarly journals Swan: a library for the analysis and visualization of long-read transcriptomes

2020 ◽  
Author(s):  
Fairlie Reese ◽  
Ali Mortazavi
Keyword(s):  
Rna Sequencing ◽  
Cell Lines ◽  
Intron Retention ◽  
Exon Skipping ◽  
Differentially Expressed ◽  
Transcript Isoforms ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Long Read

Abstract Motivation Long-read RNA-sequencing technologies such as PacBio and Oxford Nanopore have discovered an explosion of new transcript isoforms that are difficult to visually analyze using currently available tools. We introduce the Swan Python library, which is designed to analyze and visualize transcript models. Results Swan finds 4909 differentially expressed transcripts between cell lines HepG2 and HFFc6, including 279 that are differentially expressed even though the parent gene is not. Additionally, Swan discovers 285 reproducible exon skipping and 47 intron retention events not recorded in the GENCODE v29 annotation. Availability and implementation The Swan library for Python 3 is available on PyPi at https://pypi.org/project/swan-vis/ and on GitHub at https://github.com/mortazavilab/swan_vis.

scholarly journals Targeting the 16S rRNA Gene for Bacterial Identification in Complex Mixed Samples: Comparative Evaluation of Second (Illumina) and Third (Oxford Nanopore Technologies) Generation Sequencing Technologies

2019 ◽  
Vol 21 (1) ◽  
pp. 298 ◽  
Author(s):  
Raf Winand ◽  
Bert Bogaerts ◽  
Stefan Hoffman ◽  
Loïc Lefevre ◽  
Maud Delvoye ◽  
...  
Keyword(s):  
Comparative Evaluation ◽  
Species Level ◽  
Rrna Gene ◽  
Bacterial Identification ◽  
Sequencing Technologies ◽  
16S Gene ◽  
Oxford Nanopore ◽  
Oxford Nanopore Technologies ◽  
Generation Sequencing ◽  
Mixed Samples

Rapid, accurate bacterial identification in biological samples is an important task for microbiology laboratories, for which 16S rRNA gene Sanger sequencing of cultured isolates is frequently used. In contrast, next-generation sequencing does not require intermediate culturing steps and can be directly applied on communities, but its performance has not been extensively evaluated. We present a comparative evaluation of second (Illumina) and third (Oxford Nanopore Technologies (ONT)) generation sequencing technologies for 16S targeted genomics using a well-characterized reference sample. Different 16S gene regions were amplified and sequenced using the Illumina MiSeq, and analyzed with Mothur. Correct classification was variable, depending on the region amplified. Using a majority vote over all regions, most false positives could be eliminated at the genus level but not the species level. Alternatively, the entire 16S gene was amplified and sequenced using the ONT MinION, and analyzed with Mothur, EPI2ME, and GraphMap. Although >99% of reads were correctly classified at the genus level, up to ≈40% were misclassified at the species level. Both technologies, therefore, allow reliable identification of bacterial genera, but can potentially misguide identification of bacterial species, and constitute viable alternatives to Sanger sequencing for rapid analysis of mixed samples without requiring any culturing steps.

scholarly journals Picopore: A tool for reducing the storage size of Oxford Nanopore Technologies datasets without loss of functionality

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 227 ◽  
Author(s):  
Scott Gigante
Keyword(s):  
Data Storage ◽  
Data Generation ◽  
Biologically Relevant ◽  
Sequencing Technologies ◽  
Long Term Storage ◽  
Oxford Nanopore ◽  
Long Read ◽  
Oxford Nanopore Technologies ◽  
Term Storage

Oxford Nanopore Technologies' (ONT's) MinION and PromethION long-read sequencing technologies are emerging as genuine alternatives to established Next-Generation Sequencing technologies. A combination of the highly redundant file format and a rapid increase in data generation have created a significant problem both for immediate data storage on MinION-capable laptops, and for long-term storage on lab data servers. We developed Picopore, a software suite offering three methods of compression. Picopore's lossless and deep lossless methods provide a 25% and 44% average reduction in size, respectively, without removing any data from the files. Picopore's raw method provides an 88% average reduction in size, while retaining biologically relevant data for the end-user. All methods have the capacity to run in real-time in parallel to a sequencing run, reducing demand for both immediate and long-term storage space.

scholarly journals Chasing perfection: validation and polishing strategies for telomere-to-telomere genome assemblies

2021 ◽  
Author(s):  
Arang Rhie ◽  
Ann Mc Cartney ◽  
Kishwar Shafin ◽  
Michael Alonge ◽  
Andrey Bzikadze ◽  
...  
Keyword(s):  
Genome Assembly ◽  
Tandem Repeats ◽  
Hydatidiform Mole ◽  
Segmental Duplications ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Human Genome Assembly ◽  
Long Read ◽  
Genome Assemblies ◽  
Oxford Nanopore Technologies

Abstract Advances in long-read sequencing technologies and genome assembly methods have enabled the recent completion of the first Telomere-to-Telomere (T2T) human genome assembly, which resolves complex segmental duplications and large tandem repeats, including centromeric satellite arrays in a complete hydatidiform mole (CHM13). Though derived from highly accurate sequencing, evaluation revealed that the initial T2T draft assembly had evidence of small errors and structural misassemblies. To correct these errors, we designed a novel repeat-aware polishing strategy that made accurate assembly corrections in large repeats without overcorrection, ultimately fixing 51% of the existing errors and improving the assembly QV to 73.9. By comparing our results to standard automated polishing tools, we outline common polishing errors and offer practical suggestions for genome projects with limited resources. We also show how sequencing biases in both PacBio HiFi and Oxford Nanopore Technologies reads cause signature assembly errors that can be corrected with a diverse panel of sequencing technologies

scholarly journals Direct oligonucleotide sequencing with nanopores

2020 ◽  
Author(s):  
Sachin Chalapati ◽  
Conor Crosbie ◽  
Dixita Limbachiya ◽  
Nimesh Chandra Pinnamaneni
Keyword(s):  
Data Storage ◽  
Single Strand ◽  
Library Preparation ◽  
Raw Data ◽  
Base Level ◽  
Minimal Sample ◽  
Oxford Nanopore ◽  
Annealing Step ◽  
Novel Applications ◽  
Oxford Nanopore Technologies

Abstract Third-generation DNA sequencing has enabled users to sequence long, unamplified DNA fragments with minimal sample and library preparation steps. Sequencing single-stranded nucleic acids directly without amplification or by ligating a spacer strand are challenging, as the single-strand species are poor templates to add the sequencing adapters. Sequencing ssDNA or RNA directly gives valuable insights like base-level modifications and degradation levels along with saving valuable time and resources. Biological nanopores used by Oxford Nanopore Technologies process the target strands at a single-strand level, although the typical samples sequenced are double-stranded or converted into double-strand. We have identified that the MinION platform from Oxford Nanopore can perform sequencing of short, single-strand oligonucleotides directly without amplification or second-strand synthesis by performing an annealing step before library preparation. Short 5’ phosphorylated oligos when annealed to an adapter sequence can be directly sequenced in the 5' to 3' direction via nanopores, the adapters were designed to bind to the 5’ end of the oligos and leave a 3’ adenosine overhang after binding to their target. The 3’ adenosine overhang of the adapter and the terminal phosphate makes the 5’ end of the oligo to be analogous to an end-prepared dsDNA, rendering it compatible with ligation-based library preparation for sequencing. An oligo-pool containing 42,000 orthogonal sequences of 120 bp length were sequenced using the method and 37,265 of the total sequences were recovered with high accuracy. While analyzing the raw data, we had interesting observations. In our raw data, we have identified that empty signals can be wrongly identified as a valid read by the MinION platform and sometimes multiple signals containing several strands can be fused into a single read by the platforms segmentation faults. We believe that this method could enable novel applications of nanopore sequencing in DNA data-storage systems where short oligonucleotides function as the primary information carriers.

scholarly journals Construction of a chromosome-scale long-read reference genome assembly for potato

GigaScience ◽  
2020 ◽  
Vol 9 (9) ◽  
Author(s):  
Gina M Pham ◽  
John P Hamilton ◽  
Joshua C Wood ◽  
Joseph T Burke ◽  
Hainan Zhao ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Reference Genome ◽  
Agronomic Traits ◽  
Solanum Tuberosum L ◽  
Fold Increase ◽  
High Quality ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Long Read ◽  
Oxford Nanopore Technologies

Abstract Background Worldwide, the cultivated potato, Solanum tuberosum L., is the No. 1 vegetable crop and a critical food security crop. The genome sequence of DM1–3 516 R44, a doubled monoploid clone of S. tuberosum Group Phureja, was published in 2011 using a whole-genome shotgun sequencing approach with short-read sequence data. Current advanced sequencing technologies now permit generation of near-complete, high-quality chromosome-scale genome assemblies at minimal cost. Findings Here, we present an updated version of the DM1–3 516 R44 genome sequence (v6.1) using Oxford Nanopore Technologies long reads coupled with proximity-by-ligation scaffolding (Hi-C), yielding a chromosome-scale assembly. The new (v6.1) assembly represents 741.6 Mb of sequence (87.8%) of the estimated 844 Mb genome, of which 741.5 Mb is non-gapped with 731.2 Mb anchored to the 12 chromosomes. Use of Oxford Nanopore Technologies full-length complementary DNA sequencing enabled annotation of 32,917 high-confidence protein-coding genes encoding 44,851 gene models that had a significantly improved representation of conserved orthologs compared with the previous annotation. The new assembly has improved contiguity with a 595-fold increase in N50 contig size, 99% reduction in the number of contigs, a 44-fold increase in N50 scaffold size, and an LTR Assembly Index score of 13.56, placing it in the category of reference genome quality. The improved assembly also permitted annotation of the centromeres via alignment to sequencing reads derived from CENH3 nucleosomes. Conclusions Access to advanced sequencing technologies and improved software permitted generation of a high-quality, long-read, chromosome-scale assembly and improved annotation dataset for the reference genotype of potato that will facilitate research aimed at improving agronomic traits and understanding genome evolution.

Nanotrap Particles Improve Nanopore Sequencing of SARS-CoV-2 and Other Respiratory Viruses

2021 ◽  
Author(s):  
Patrick Daniel Andersen ◽  
Stephanie Barksdale ◽  
Robert Alex Barclay ◽  
Natalie Smith ◽  
Justin Fernandes ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Nanopore Sequencing ◽  
Sequencing Platform ◽  
Magnetic Hydrogel ◽  
Oxford Nanopore ◽  
Input Sample ◽  
Lower Titer ◽  
Syncytial Virus ◽  
Hydrogel Particle ◽  
Oxford Nanopore Technologies

Presented here is a magnetic hydrogel particle enabled workflow for capturing and concentrating SARS-CoV-2 from diagnostic remnant swab samples that significantly improves sequencing results using the Oxford Nanopore Technologies MinION sequencing platform. Our approach utilizes a novel affinity-based magnetic hydrogel particle, circumventing low input sample volumes and allowing for both rapid manual and automated high throughput workflows that are compatible with nanopore sequencing. This approach enhances standard RNA extraction protocols, providing up to 40x improvements in viral mapped reads, and improves sequencing coverage by 20-80% from lower titer diagnostic remnant samples. Furthermore, we demonstrate that this approach works for contrived influenza virus and respiratory syncytial virus samples, suggesting that it can be used to identify and improve sequencing results of multiple viruses in VTM samples. These methods can be performed manually or on a KingFisher Apex system.

Sign in / Sign up

Export Citation Format

Share Document