Benchmarking of the Oxford Nanopore MinION Sequencing for Quantitative and Qualitative Assessment of CDNA Populations

Overview

Journal Sci Rep

Specialty Science

Date 2016 Aug 25

PMID 27554526

Citations 78

Authors

Spyros Oikonomopoulos

Yu Chang Wang

Haig Djambazian

Dunarel Badescu

Jiannis Ragoussis

Affiliations

Soon will be listed here.

Abstract

To assess the performance of the Oxford Nanopore Technologies MinION sequencing platform, cDNAs from the External RNA Controls Consortium (ERCC) RNA Spike-In mix were sequenced. This mix mimics mammalian mRNA species and consists of 92 polyadenylated transcripts with known concentration. cDNA libraries were generated using a template switching protocol to facilitate the direct comparison between different sequencing platforms. The MinION performance was assessed for its ability to sequence the cDNAs directly with good accuracy in terms of abundance and full length. The abundance of the ERCC cDNA molecules sequenced by MinION agreed with their expected concentration. No length or GC content bias was observed. The majority of cDNAs were sequenced as full length. Additionally, a complex cDNA population derived from a human HEK-293 cell line was sequenced on an Illumina HiSeq 2500, PacBio RS II and ONT MinION platforms. We observed that there was a good agreement in the measured cDNA abundance between PacBio RS II and ONT MinION (rpearson = 0.82, isoforms with length more than 700bp) and between Illumina HiSeq 2500 and ONT MinION (rpearson = 0.75). This indicates that the ONT MinION can sequence quantitatively both long and short full length cDNA molecules.

Citing Articles

Germline mosaicism in TCF20-associated neurodevelopmental disorders: a case study and literature review.

Poquerusse J, Whitford W, Taylor J, Gregersen N, Love D, Tsang B J Hum Genet. 2025; 70(4):215-222.

PMID: 40011607 PMC: 11882450. DOI: 10.1038/s10038-025-01323-3.

Adaptable and comprehensive approaches for long-read nanopore sequencing of polyadenylated and non-polyadenylated RNAs.

Haile S, Corbett R, ONeill K, Xu J, Smailus D, Pandoh P Front Genet. 2024; 15:1466338.

PMID: 39687742 PMC: 11647301. DOI: 10.3389/fgene.2024.1466338.

Real-time genomic characterization of pediatric acute leukemia using adaptive sampling.

Geyer J, Opoku K, Opoku K, Lin J, Ramkissoon L, Mullighan C bioRxiv. 2024; .

PMID: 39416119 PMC: 11483067. DOI: 10.1101/2024.10.11.617690.

Detection of O157:H7 in Ground Beef Using Long-Read Sequencing.

Counihan K, Kanrar S, Tilman S, Capobianco J, Armstrong C, Gehring A Foods. 2024; 13(6).

PMID: 38540819 PMC: 10969500. DOI: 10.3390/foods13060828.

PHARE: a bioinformatics pipeline for compositional profiling of multiclonal Plasmodium falciparum infections from long-read Nanopore sequencing data.

Hosch S, Wagner P, Giger J, Dubach N, Saavedra E, Perno C J Antimicrob Chemother. 2024; 79(5):987-996.

PMID: 38502783 PMC: 11062946. DOI: 10.1093/jac/dkae060.

References

Au K, Sebastiano V, Afshar P, Durruthy J, Lee L, Williams B . Characterization of the human ESC transcriptome by hybrid sequencing. Proc Natl Acad Sci U S A. 2013; 110(50):E4821-30. PMC: 3864310. DOI: 10.1073/pnas.1320101110. View

Picelli S, Faridani O, Bjorklund A, Winberg G, Sagasser S, Sandberg R . Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc. 2014; 9(1):171-81. DOI: 10.1038/nprot.2014.006. View

Quick J, Quinlan A, Loman N . A reference bacterial genome dataset generated on the MinION™ portable single-molecule nanopore sequencer. Gigascience. 2014; 3:22. PMC: 4226419. DOI: 10.1186/2047-217X-3-22. View

Trapnell C, Williams B, Pertea G, Mortazavi A, Kwan G, van Baren M . Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 2010; 28(5):511-5. PMC: 3146043. DOI: 10.1038/nbt.1621. View

Dabney J, Meyer M . Length and GC-biases during sequencing library amplification: a comparison of various polymerase-buffer systems with ancient and modern DNA sequencing libraries. Biotechniques. 2012; 52(2):87-94. DOI: 10.2144/000113809. View

Marinov G, Williams B, McCue K, Schroth G, Gertz J, Myers R . From single-cell to cell-pool transcriptomes: stochasticity in gene expression and RNA splicing. Genome Res. 2013; 24(3):496-510. PMC: 3941114. DOI: 10.1101/gr.161034.113. View

Stein A, Takasuka T, Collings C . Are nucleosome positions in vivo primarily determined by histone-DNA sequence preferences?. Nucleic Acids Res. 2009; 38(3):709-19. PMC: 2817465. DOI: 10.1093/nar/gkp1043. View

Patro R, Mount S, Kingsford C . Sailfish enables alignment-free isoform quantification from RNA-seq reads using lightweight algorithms. Nat Biotechnol. 2014; 32(5):462-4. PMC: 4077321. DOI: 10.1038/nbt.2862. View

Quail M, Smith M, Coupland P, Otto T, Harris S, Connor T . A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers. BMC Genomics. 2012; 13:341. PMC: 3431227. DOI: 10.1186/1471-2164-13-341. View

10.

Zhulidov P, Bogdanova E, Shcheglov A, Vagner L, Khaspekov G, Kozhemyako V . Simple cDNA normalization using kamchatka crab duplex-specific nuclease. Nucleic Acids Res. 2004; 32(3):e37. PMC: 373426. DOI: 10.1093/nar/gnh031. View

11.

Rothberg J, Hinz W, Rearick T, Schultz J, Mileski W, Davey M . An integrated semiconductor device enabling non-optical genome sequencing. Nature. 2011; 475(7356):348-52. DOI: 10.1038/nature10242. View

12.

Eid J, Fehr A, Gray J, Luong K, Lyle J, Otto G . Real-time DNA sequencing from single polymerase molecules. Science. 2008; 323(5910):133-8. DOI: 10.1126/science.1162986. View

13.

Bray N, Pimentel H, Melsted P, Pachter L . Near-optimal probabilistic RNA-seq quantification. Nat Biotechnol. 2016; 34(5):525-7. DOI: 10.1038/nbt.3519. View

14.

Ferrarini M, Moretto M, Ward J, Surbanovski N, Stevanovic V, Giongo L . An evaluation of the PacBio RS platform for sequencing and de novo assembly of a chloroplast genome. BMC Genomics. 2013; 14:670. PMC: 3853357. DOI: 10.1186/1471-2164-14-670. View

15.

Angelini C, De Canditiis D, De Feis I . Computational approaches for isoform detection and estimation: good and bad news. BMC Bioinformatics. 2014; 15:135. PMC: 4098781. DOI: 10.1186/1471-2105-15-135. View

16.

Pertea M, Pertea G, Antonescu C, Chang T, Mendell J, Salzberg S . StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat Biotechnol. 2015; 33(3):290-5. PMC: 4643835. DOI: 10.1038/nbt.3122. View

17.

Benitez-Paez A, Portune K, Sanz Y . Species-level resolution of 16S rRNA gene amplicons sequenced through the MinION™ portable nanopore sequencer. Gigascience. 2016; 5:4. PMC: 4730766. DOI: 10.1186/s13742-016-0111-z. View

18.

Pelechano V, Wei W, Steinmetz L . Extensive transcriptional heterogeneity revealed by isoform profiling. Nature. 2013; 497(7447):127-31. PMC: 3705217. DOI: 10.1038/nature12121. View

19.

Bentley D, Balasubramanian S, Swerdlow H, Smith G, Milton J, Brown C . Accurate whole human genome sequencing using reversible terminator chemistry. Nature. 2008; 456(7218):53-9. PMC: 2581791. DOI: 10.1038/nature07517. View

20.

Zhu Y, Machleder E, Chenchik A, Li R, Siebert P . Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction. Biotechniques. 2001; 30(4):892-7. DOI: 10.2144/01304pf02. View