Comprehensive comparison of Pacific Biosciences and Oxford Nanopore Technologies and their applications to transcriptome analysis

Abstract

Background: Given the demonstrated utility of Third Generation Sequencing [Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT)] long reads in many studies, a comprehensive analysis and comparison of their data quality and applications is in high demand. Methods: Based on the transcriptome sequencing data from human embryonic stem cells, we analyzed multiple data features of PacBio and ONT, including error pattern, length, mappability and technical improvements over previous platforms. We also evaluated their application to transcriptome analyses, such as isoform identification and quantification and characterization of transcriptome complexity, by comparing the performance of size-selected PacBio, non-size-selected ONT and their corresponding Hybrid-Seq strategies (PacBio+Illumina and ONT+Illumina). Results: PacBio shows overall better data quality, while ONT provides a higher yield. As with data quality, PacBio performs marginally better than ONT in most aspects for both long reads only and Hybrid-Seq strategies in transcriptome analysis. In addition, Hybrid-Seq shows superior performance over long reads only in most transcriptome analyses. Conclusions: Both PacBio and ONT sequencing are suitable for full-length single-molecule transcriptome analysis. As this first use of ONT reads in a Hybrid-Seq analysis has shown, both PacBio and ONT can benefit from a combined Illumina strategy. The tools and analytical methods developed here provide a resource for future applications and evaluations of these rapidly-changing technologies.

A case of genotype‐3b hepatitis C virus in which the whole genome was successfully analyzed using third‐generation nanopore sequencing

“…In the present analysis, the insertion and deletion rates were 2.9% and 6.0%, respectively, and both values are nearly equivalent to those previously reported, 2.9% and 7.5%. 23 Thus, the accuracy of nanopore sequencing is insufficient for deep sequencing, and we applied this system instead of direct sequencing of genomes in which primers for sequencing have not yet been determined; the sequence of the HCV genome was determined based on the major nucleotide in each nucleotide position, excluding minor nucleotides that might have resulted from sequencing errors.…”

Going the Distance: Optimizing RNA-Seq Strategies for Transcriptomic Analysis of Complex Viral Genomes

“…Both approaches are currently highly constrained by the amount of starting material required (generally Ͼ500 ng of polyadenylated RNA) and produce comparatively few reads (less than one million reads per run), which limits the depth of sequencing. Likewise, both suffer from high error rates (47), although these are lower for SMRT sequencing, which also benefits from the dual capture of the 5= cap and 3= poly(A) tail, enabling the accurate mapping of transcription start and RNA cleavage sites. These advantages must be offset against a more involved sequencing protocol, which includes reverse transcription and PCR steps and the need to size-select fragments prior to sequencing.…”

“…Although a single nanopore MinION run can generate upwards of double the number of reads as SMRT sequencing, the error rate is, currently, notably higher (47). The accurate mapping of sequence reads first requires error correction, a complex proposition, to accurately map the extreme 5= and 3= ends of transcripts, as well as accurately identify sites of splicing.…”

Rapid, Unbiased PRRSV Strain Detection Using MinION Direct RNA Sequencing and Bioinformatics Tools

“…Total yield, total reads, read quality, and read length from whole genome sequencing were analyzed using NanoPlot [49]. To obtain raw error rates and error patterns, sequencing reads were mapped to the VR2332 reference sequence using minimap2 [50], processed with SAMtools [51] to generate BAM files, and then evaluated by AlignQC [52].…”