The anvi'o 'trnaseq' workflow

Process transfer RNA transcripts from tRNA-seq datasets

The trnaseq workflow takes in raw paired-end sequencing data generated from trna-seq libraries (i.e., the direct sequencing of transfer RNA transcripts from cultures or environmental samples), and processes these data to identify tRNA sequences and their structural features, predict chemical modification sites and modification fractions across samples, assign taxonomy to tRNA transcript seeds, and generate tables and summary data for downstream analyses. The tRNA-seq resources in anvi'o are operational, however, they are experimental. If you have datasets that are suitable for analysis, pelase consider getting in touch with us first.

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Authors

Artifacts accepted

The trnaseq can typically be initiated with the following artifacts:

workflow-config samples-txt

Artifacts produced

The trnaseq typically produce the following anvi’o artifacts:

trnaseq-db trnaseq-contigs-db trnaseq-profile-db trnaseq-seed-txt modifications-txt

Third party programs

This is a list of programs that may be used by the trnaseq workflow depending on the user settings in the workflow-config :

An anvi’o installation that follows the recommendations on the installation page will include all these programs. But please consider your settings, and cite these additional tools from your methods sections.

Workflow description and usage

The tRNA-seq workflow is a Snakemake workflow run by anvi-run-workflow.

The workflow can run the following programs in order:

Input

The tRNA-seq workflow requires two files to run: a workflow-config config file and a samples-txt. You can obtain a ‘default’ config file for this workflow to further edit using the following command.

anvi-run-workflow -w trnaseq \ --get-default-config config.json

Different “rules,” or steps, of the workflow can be turned on and off as needed in the config file. The workflow can be restarted at intermediate rules without rerunning prior rules that have already completed.

samples-txt will contain a list of FASTQ or FASTA files and associated information on each library. FASTQ files contain unmerged paired-end tRNA-seq reads. Reads are merged in the workflow by Illumina-utils. FASTA files contain merged reads, and the initial read-merging steps in the workflow are skipped.

Here is an example tRNA-seq samples file with FASTQ inputs.

sample treatment r1 r2 r1_prefix r2_prefix
ecoli_A1_noDM untreated FASTQ/ecoli_A1_noDM.r1.fq.gz FASTQ/ecoli_A1_noDM.r2.fq.gz NNNNNN TTCCAGT
ecoli_A1_DM demethylase FASTQ/ecoli_A1_DM.r1.fq.gz FASTQ/ecoli_A1_DM.r2.fq.gz NNNNNN TCTGAGT
ecoli_B1_noDM untreated FASTQ/ecoli_B1_noDM.r1.fq.gz FASTQ/ecoli_B1_noDM.r2.fq.gz NNNNNN TGGTAGT
ecoli_B1_DM demethylase FASTQ/ecoli_B1_DM.r1.fq.gz FASTQ/ecoli_B1_DM.r2.fq.gz NNNNNN CTGAAGT

The treatment column is optional. The treatment indicates a chemical application, such as demethylase, and can be used to have a bearing on seed sequence determination in anvi-merge-trnaseq. In the absence of a treatment column, all samples are assigned the same treatment, which can be specified in the anvi_trnaseq section of the workflow config file and defaults to untreated.

Read 1 and 2 prefix columns are also optional. These represent sequences that Illumina-utils should identify and trim from the start of the read. In the example, the read 1 prefix is a unique molecular identifier (UMI) of 6 random nucleotides, and the read 2 prefix is a sample barcode. Illumina-utils will discard the paired-end read if the prefix is not found. In the example, the read 1 UMI will always be found, but the read 2 barcode must match exactly.

Here is an equivalent tRNA-seq samples file with FASTA inputs.

sample treatment fasta
ecoli_A1_noDM untreated FASTA/ecoli_A1_noDM.fa.gz
ecoli_A1_DM demethylase FASTA/ecoli_A1_DM.fa.gz
ecoli_B1_noDM untreated FASTA/ecoli_B1_noDM.fa.gz
ecoli_B1_DM demethylase FASTA/ecoli_B1_DM.fa.gz

Note that barcodes and other sequence prefixes should already be trimmed from FASTA sequences.

Edit this file to update this information.