@@ -24,4 +24,6 @@ Variant calling analysis on RNA-Seq base editing data:
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@@ -24,4 +24,6 @@ Variant calling analysis on RNA-Seq base editing data:
- Mark duplicates with _Picard MarkDuplicates_ and split of eads containing Ns with _GATK SplitNCigarReads_
- Mark duplicates with _Picard MarkDuplicates_ and split of eads containing Ns with _GATK SplitNCigarReads_
- Variant calling using three different tools: _HaplotypeCaller_ (with options `--min-base-quality-score 20`, `--dont-use-soft-clipped-bases`, and `–standard-min-confidence-threshold-for-calling 20`), _Mutect2_ (in tumor-only mode, with options `--disable-read-filter MateOnSameContigOrNoMappedMateReadFilter`), and _FreeBayes_.
- Variant calling using three different tools: _HaplotypeCaller_ (with options `--min-base-quality-score 20`, `--dont-use-soft-clipped-bases`, and `–standard-min-confidence-threshold-for-calling 20`), _Mutect2_ (in tumor-only mode, with options `--disable-read-filter MateOnSameContigOrNoMappedMateReadFilter`), and _FreeBayes_.
Nucleotide composition of each position was also assessed using REDItools (https://github.com/tflati/reditools2.0) on each sample, discarding all the positions having coverage lower than 20 and base quality lower than 30 to avoid errors due to low sampling. Next, variants called by each tool in the untreated controls were filtered out in the treated samples to enrich for private mutations. This procedure retained only variants in high-quality genomic positions in both treated and untreated sample, for which the untreated sample showed ≥ 99% of reads supporting the reference, non-mutant, base at the position of the mutation (based on REDItools). The final lists of variants for each sample were made by those called by all tools and passing the filtering procedure (intersection).
Nucleotide composition of each position was also assessed using [_REDItools_](https://github.com/tflati/reditools2.0) on each sample, discarding all the positions having coverage lower than 20 and base quality lower than 30 to avoid errors due to low sampling.</br>
Variants called by each tool in the untreated controls were filtered out in the treated samples to enrich for private mutations. This procedure retained only variants in high-quality genomic positions in both treated and untreated sample, for which the untreated sample showed ≥ 99% of reads supporting the reference, non-mutant, base at the position of the mutation (based on REDItools).</br>
The final lists of variants for each sample were made by those called by all tools and passing the filtering procedure (intersection).
