Direct Nanopore Sequencing of Individual Full Length tRNA Strands

We describe a method for direct tRNA sequencing using the Oxford Nanopore MinION. The principal technical advance is custom adapters that facilitate end-to-end sequencing of individual tRNA molecules. A second advance is a Nanopore sequencing pipeline optimized for tRNA. We tested this method using purified E. coli tRNAfMet, tRNALys, and tRNAPhe samples. 76-to-92% of individual aligned tRNA sequence reads were full length. As proof of concept, we showed that Nanopore sequencing detected all 42 expected tRNA isoacceptors in total E. coli tRNA. Alignment-based comparison between the three purified tRNAs and their synthetic controls revealed systematic miscalls at or adjacent to the positions of known nucleotide modifications. Systematic miscalls were also observed proximal to known modifications in total E. coli tRNA alignments.

Direct-seq: employing programmed gRNA scaffold for streamlined scRNA-seq in CRISPR screen

This protocol aimed to combine the single cell RNA-seq (scRNA-seq) with the CRISPR screening assay based on 10x 3’ RNA-seq platform to profile the transcriptome together with genotype at single cell resolution. To achieve this goal, an A/G mixed capture sequence and tRNA sequence are incorporated into the Tetraloop, Loop2 and Tail of the gRNA scaffold. Therefore, cDNA derived from the gRNA transcripts can be captured and barcoded by poly(dT) primer together with the endogenous mRNA and enriched by the nest PCR. With this method, the CRISPR perturbation and its transcriptional readouts can be profiled together in a streamlined workflow. This method, which we named as Direct-seq, enabled the direct genotyping and expression profiling of the CRISPR screening products at single cell resolution on versatile scRNA-seq platforms across different throughput, including 10x 3’ and 5’ RNA-seq, Fluidigim C1, SMART-seq, BGI DNBelab C4 and others.

tRNAviz: explore and visualize tRNA sequence features

Transfer RNAs (tRNAs) are ubiquitous across the tree of life. Although tRNA structure is highly conserved, there is still significant variation in sequence features between clades, isotypes and even isodecoders. This variation not only impacts translation, but as shown by a variety of recent studies, nontranslation-associated functions are also sensitive to small changes in tRNA sequence. Despite the rapidly growing number of sequenced genomes, there is a lack of tools for both small- and large-scale comparative genomics analysis of tRNA sequence features. Here, we have integrated over 150 000 tRNAs spanning all domains of life into tRNAviz, a web application for exploring and visualizing tRNA sequence features. tRNAviz implements a framework for determining consensus sequence features and can generate sequence feature distributions by isotypes, clades and anticodons, among other tRNA properties such as score. All visualizations are interactive and exportable. The web server is publicly available at http://trna.ucsc.edu/tRNAviz/.

Genomic Sequence of Mycobacteriophage OKCentral2016

ABSTRACT OKCentral2016 is the first mycobacteriophage sequenced from Oklahoma soil using the bacterial host Mycobacterium smegmatis strain mc 2 155. OKCentral2016 has a double-stranded DNA genome composed of 50,072 bp, with 84 predicted coding genes and 1 tRNA sequence. This mycobacteriophage has sequence similarities to members of the A10 subcluster.

Strand transfer to the 5′ part of a tRNA as a mechanism for retrovirus patch-repair recombination in vivo

By screening for marker-cassette deletion mutants of a murine leukaemia virus-based replication-competent vector, two occurrences of tRNA sequence patch insertions were identified. In one of the cases, 28 nucleotides from the 5′ end of tRNALys4 were inserted in the plus-strand orientation, which points to a novel strand-transfer mechanism to tRNAs during reverse transcriptase-mediated retroviral recombination.