Updates and bugfixes

Updates

• Added Biopython SeqIO functionality for Stockholm file parsing - see ssAlign.py. This removes all necessity to filter tRNAs with poor secondary structure alignment from the input reference. This was being done before for some genes in hg38 and gorGor4 references to prevent multi-line alignments in stk files.
• Updated gorGor4 and hg38 references. No genes are filtered out from full set (except for one gene in gorGor4, see README
• Added C. elegans reference and updates to A. thaliana reference
• Misincorporation signature required to find new modification was previously set at no more then 95% misincorporation of one nucleotide (to distinguish from SNPs or inosine). This is now 97% which we find more permissive to detect important modified sites that are verified as modifications.

Bugfixes

• DESeq2 dot plot axis labels: remove "log10" as the values are not log scaled but only the scale of the axis.
• Installation instructions updated to include the installation and use of condaforge/mambaforge offering better speed and environment resolution and dependency handling.

New SLAC functionality

Updated new functionality for SingLe-read Analysis of Crosstalks (SLAC)

• This analysis allows the detection of transcriptome-wide crosstalks between pairs of tRNA modifications and modification-aminoacylation.
• Implemented using --crosstalks parameter
• See details of analysis at https://mim-trnaseq.readthedocs.io/en/latest/intro.html and details of outputs at https://mim-trnaseq.readthedocs.io/en/latest/output.html

Bugfixes

• New mods discovery for mitochondrial sequences is now agnostic to coverage at positions in these references. Often, the coverage of mito tRNAs is much lower than their cytosolic counterparts, which makes new mod discovery here difficult when there is a minimum nucleotide coverage filter. This is now disabled.

Minor bugfixes - plastid tRNAs, tRX cluster parents, DESeq heatmaps

Bugfixes

• When specifying custom input fasta sequences, plastid tRNAs are now specified as a separate file and handled independently from mitochondrial sequences
• If a cluster with a "tRX" parent sequence is unsplit from a "tRNA" child, naming conventions for this composite cluster are now correct
• When a heatmap of differentially expressed isodecoders or anticodons only has one sequence, the "basemean" for the sequence in the right most pane of the heatmap is given as a bar plot, not a line plot. Avoids errors in trying to make line plots from one data point.

Minor update to outputs

New output

• modPos_totalMisincProp.csv output now saved to /mods directory. This contains total misincorporation proportions (opposed to misincoporation rates for each of the 4 possible nucleotides, as in mismatchTable.csv) per transcript at conserved modification sites, as well as the identity of the nucleotide at that position and coverage info.

Minor bugfix 1.1.6

Bugfix

• --min-cov now filters unique transcripts from coverage, mismatch, readthrough and stop tables where the proportional coverage does not meet the --min-cov threshold in ALL samples. That is, if ANY sample meets the threshold, the transcript is not filtered. Previously, transcripts not meeting the threshold in any sample were filtered, sometimes leading to transcripts with significant regulation in one or more condition being removed from these tables.

STAR Protocols mim-tRNAseq

v1.1.5-alpha

version bump

New release v1.1.5

Updates

• Added mitochondrial in addition to plastid sequences for TAIR10 (Arabidopsis thaliana)
• New code to handle both mito and plastid sequences together for plant species
• New colourblind friendly purple/green colour scale for DESeq plots (dot plots and heatmaps) and differential modification heatmaps

Bugfixes

• Fixed isodecoder size in *isodecoderInfo.txt for single transcript clusters
• Prevent overwriting of normalised counts files for cyto and organelle in /counts folder. Distinct naming for separate files.

v1.1

Updates

• DESeq2 and differential modification analysis now also run separately on organellar tRNAs (mitochondrial or plastid)
• New extended documentation (https://mim-trnaseq.readthedocs.io/)
• mm39 Mouse reference added and replaced old mm10 reference. --species Mmus will specify new mm39 reference
• Arabidopsis thalina TAIR10 reference added to prebuilt references and available in --species flag
• Renaming of plastid tRNA genes in output (i.e. no "mito" but "plastid" instead)

Bugfixes

• New mods detection was excluding all A nucleotides from detection! These are now detectable and written to predictedMods.csv again
• Fixed errors when clustering is disabled
• Automatic checking and error handling for duplicated fastq inputs in sample data file
• Fixed bug for normalised count output from DESeq2 for single replicate conditions
• Handling of clusters with non-deconvoluted mixed tRNA and tRX genes; these composite clusters retain indication of the tRX genes e.g. tRNA-Ala-AGC-1/2/tRX5

Stable version v1.0

Release v1.0 contains major upgrades to deconvolution and other optimisations and bug fixes

Deconvolution

Clusters that cannot be deconvoluted

• New deconvolution algorithm with updated filtering for clusters that cannot be deconvoluted

1. 3’ coverage bias at a mismatch required for deconvolution. In order to overcome this, the –deconv-cov-ratio parameter can be used to set a threshold for the difference in coverage at mismatch and 3' end of tRNA. Sequences not passing this threshold will be marked as not deconvoluted.
2. Some tRNAs may only be distinguishable from the parent cluster by positions that might also be modified sites. Such tRNA transcripts (and the parent of the cluster) are also labelled as not deconvoluted.
3. Reads that cannot be assigned to a transcript within a cluster are assumed to originate from the parent and are, by default, left assigned to the parent sequence. However, in some cases these parent-assigned reads also contain erroneous mismatches which indicate that they might not indeed belong to the parent transcript either. If 10% or more parent-assigned reads contain such mismatches, the entire cluster is not deconvoluted as these reads are likely to significantly impact the correct estimation of parent abundance, and can also impact the deconvolution choice made my mim-tRNAseq for other members of the cluster.

New handling and naming on unsplit clusters

Transcripts that are not deconvoluted are renamed to provide details on which transcripts remain clustered. These are then treated as other single transcripts for differential expression analysis, modification profiling, and other downstream analyses. For example, if Ala-AGC-1 and Ala-AGC-2 are clustered and cannot be deconvoluted, these two transcripts will remain clustered, be renamed to Ala-AGC1/2 (the parent isodecoder number for the cluster is always listed first), and appear as a single entry for counts, modification analysis, differential expression, and coverage data. For the purpose of readability in plots, the naming of unsplit clusters is shown as Ala-AGC-1-multi. This is particularly useful for clusters with many unsplit sequences as these have unnecessarily long labels for display purposes.

New output: annotation/*_unsplitClusterInfo.txt

This new output details the clusters that were not split as described above, the number of transcripts, the parent of the cluster and the reason the cluster could not be split (see output documentation)

Minor updates and bug fixes

• Fixed tie-breaking algorithm for assigning reads during deconvolution when there are potential ties in the choice
• Fixed erroneous new modification detection at position 34
• Added rat rn7 reference

DESeq2 adjusted p-value threshold and minor bugfixes

New features and minor bugfixes

Adjusted p-value threshold (#14)

• Users can now set an adjusted p-value threshold for plots generated from DESeq2 differential expression analysis. Default is adj. p-value <= 0.05.
• Adj. p-value now also displayed on all *-diffexpr-countplot.pdf files

Bugfixes

• knownMods table now a defaultdict(list) to prevent KeyError after 1st mapping round and subtracting predicted from known mods in unknownMods() - (#9)