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

Abstract

Given the demonstrated utility of Third Generation Sequencing Background: [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.Based on the Methods: 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 PacBio, ONT and their corresponding Hybrid-Seq strategies (PacBio+Illumina and ONT+Illumina).PacBio shows overall better data quality, while ONT provides a higher Results: 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.Both PacBio and ONT Conclusions: 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

“…Thus, the read depths could be reduced because of a tendency to generate insertions and/or deletions of bases. 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% . 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.…”

Long‐Read RNA Sequencing Identifies Alternative Splice Variants in Hepatocellular Carcinoma and Tumor‐Specific Isoforms

“…Template libraries were sequenced on a Pacific Biosciences RS II sequencer using the DNA Sequencing Kit (Pacific Biosciences). Basic quality information of PacBio SMRT‐seq data was obtained by aligning long reads to human reference genome (hg19) with Genomic Mapping and Alignment Program and examined for quality by AlignQC against reference transcriptome of RefSeq gene annotation (Supporting Table ).…”

Full gene HLA class I sequences of 79 novel and 519 mostly uncommon alleles from a large United States registry population

“…In these cases, assignments are made because the current allele sequence database does not include any genotypes that require these unphased regions to be linked. Alternative strategies using sequencing of single DNA molecules with a read length over 1500 bp (ie, third‐generation sequencing) can address this issue but the higher error rate of these strategies presents another challenge . Some laboratories have adopted a hybrid strategy of combining a massive parallel sequencing platform with a third‐generation platform …”