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High-throughput total nucleic acid (TNA) purification methods based on solid-phase reversible immobilization (SPRI) beads produce TNA suitable for both genomic and transcriptomic applications. Even so, small RNA species, including miRNA, bind weakly to SPRI beads under standard TNA purification conditions, necessitating a separate workflow using column-based methods that are difficult to automate. Here, an SPRI-based high-throughput TNA purification protocol that recovers DNA, RNA and small RNA, called GSC-modified RLT+ Aline bead-based protocol (GRAB-ALL), which incorporates modifications to enhance small RNA recovery is presented. GRAB-ALL was benchmarked against existing nucleic acid purification workflows and GRAB-ALL efficiently purifies TNA, including small RNA, for next-generation sequencing applications in a plate-based format suitable for automated high-throughput sample preparation.

The analysis of cell-free circulating tumor DNA (ctDNA) is potentially a less invasive, more dynamic assessment of cancer progression and treatment response than characterizing solid tumor biopsies. Standard isolation methods require separation of plasma by centrifugation, a time-consuming step that complicates automation. To address these limitations, we present an automatable magnetic bead-based ctDNA isolation method that eliminates centrifugation to purify ctDNA directly from peripheral blood (PB). To develop and test our method, ctDNA from cancer patients was purified from PB and plasma. We found that allelic fractions of somatic single-nucleotide variants from target gene capture libraries were comparable, indicating that the PB ctDNA purification method may be a suitable replacement for the plasma-based protocols currently in use.

Pink salmon (Oncorhynchus gorbuscha) adults are the smallest of the five Pacific salmon native to the western Pacific Ocean. Pink salmon are also the most abundant of these species and account for a large proportion of the commercial value of the salmon fishery worldwide. A two-year life history of pink salmon generates temporally isolated populations that spawn either in even-years or odd-years. To uncover the influence of this genetic isolation, reference genome assemblies were generated for each year-class and whole genome re-sequencing data was collected from salmon of both year-classes. The salmon were sampled from six Canadian rivers and one Japanese river. At multiple centromeres we identified peaks of Fst between year-classes that were millions of base-pairs long. The largest Fst peak was also associated with a million base-pair chromosomal polymorphism found in the odd-year genome near a centromere. These Fst peaks may be the result of a centromere drive or a combination of reduced recombination and genetic drift, and they could influence speciation. Other regions of the genome influenced by odd-year and even-year temporal isolation and tentatively under selection were mostly associated with genes related to immune function, organ development/maintenance, and behaviour.

The advent of long-read (LR) sequencing technologies has provided a direct opportunity to determine the structure of transcripts with potential for end-to-end sequencing of full-length RNAs. LR methods that have been described to date include commercial offerings from Oxford Nanopore Technologies (ONT) and Pacific Biosciences. These kits are based on selection of polyadenylated (polyA+) RNAs and/or oligo-dT priming of reverse transcription. Thus, these approaches do not allow comprehensive interrogation of the transcriptome due to their exclusion of non-polyadenylated (polyA-) RNAs. In addition, polyA + specificity also results in 3′-biased measurements of PolyA+ RNAs especially when the RNA input is partially degraded. To address these limitations of current LR protocols, we modified rRNA depletion protocols that have been used in short-read sequencing: one approach representing a ligation-based method and the other a template-switch cDNA synthesis-based method to append ONT-specific adaptor sequences and by removing any deliberate fragmentation/shearing of RNA/cDNA. Here, we present comparisons with poly+ RNA-specific versions of the two approaches including the ONT PCR-cDNA Barcoding kit. The rRNA depletion protocols displayed higher proportions (30%–50%) of intronic content compared to that of the polyA-specific protocols (5%–8%). In addition, the rRNA depletion protocols enabled ∼20–50% higher detection of expressed genes. Other metrics that were favourable to the rRNA depletion protocols include better coverage of long transcripts, and higher accuracy and reproducibility of expression measurements. Overall, these results indicate that the rRNA depletion-based protocols described here allow the comprehensive characterization of polyadenylated and non-polyadenylated RNAs. While the resulting reads are long enough to help decipher transcript structures, future endeavors are warranted to improve the proportion of individual reads representing end-to-end spanning of transcripts.