We enjoy a good technology evaluation as much as the next scientist, particularly when it comes to sequencing. So we were quite interested in a recent F1000Research publication about long-read sequencing platforms from researchers at the University of Iowa, the University of Oxford, and other institutions.
From senior author Kin Fai Au and collaborators, “Comprehensive comparison of Pacific Biosciences and Oxford Nanopore Technologies and their applications to transcriptome analysis” presents a nice assessment of the pros and cons of long-read sequencing tools. The authors note that PacBio libraries were prepared using SageELF size selection, while the Oxford Nanopore libraries were not size-selected. Some of the study results can be explained by the difference in sample prep.
To compare the technologies, scientists sequenced the transcriptomes of human embryonic stem cells with PacBio, ONT, and Illumina (short reads were used for comparison purposes as well as for building hybrid assemblies). They note that long reads have been especially informative for transcriptome studies, including gene isoform identification.
In this analysis, both platforms were able “to provide precise and complete isoform identification” for a small library of known spike-in variants and for more complex transcriptomes as well. “PacBio has a slightly better overall performance, such as discovery of transcriptome complexity and sensitive identification of isoforms,” the team reports.
Delving into details, the long-read platforms performed similarly in read length, based on a comparison of mappable length. The team found that a higher proportion of PacBio reads could be aligned to reference genomes compared to ONT reads. Throughput was also noticeably different: “the yield per flow cell of ONT is much higher than PacBio, because each nanopore can sequence multiple molecules, while the wells of PacBio SMRT cells are not reusable,” the authors note. Error rate was another area of divergent results. PacBio CCS reads had an error rate “as low as 1.72%,” the scientists report, giving data from that platform “higher base quality than corresponding ONT data.”
“PacBio can generate extremely-low-error-rate data for high-resolution studies, which is not feasible for ONT,” the scientists add, noting that ONT advantages include high throughput and lower expense. “The cost for our ONT data generation was 1,000–2,000USD,” they report.
In addition, the scientists assessed hybrid approaches for both platforms, adding short Illumina reads for error correction. It was the first known pairing of ONT and Illumina reads for this purpose. “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 team writes. The authors note that both long-read sequencing tools have improved significantly with recent models and predict that future enhancements will be a boon to transcriptome studies as well.