Appendix: Parameters of external tools

This page lists the parameters (command-line options) that cgpRna (version: 2.3.0) uses when running external tools below.

Mapping and gene counting

• Convert BAM file to FastQ files if the input file is a BAM using bamtofastq (biobambam2 version 2.0.87)

   bamtofastq \
   exclude=QCFAIL,SECONDARY,SUPPLEMENTARY \
   gz=1 \
   level=1 \
   T=${TEMPORARY_FILE} \
   S=${SINGLE_END_FILE} \
   O=${UNMATCHED_PAIR_FIRST_MATE_FILE} \
   O2=${UNMATCHED_PAIR_SECOND_MATE_FILE} \
   F=${MATCHED_PAIR_FIRST_MATE_FILE} \
   F2=${MATCHED_PAIR_SECOND_MATE_FILE} \
   filename=${INPUT_BAM}

• Map with STAR (Version 2.5.0c)

   STAR --runMode alignReads \
   --sjdbOverhang 99 \
   --limitBAMsortRAM 64606632121 \
   --limitSjdbInsertNsj 1000000 \
   --outSAMtype BAM Unsorted \
   --outSAMstrandField intronMotif \
   --outSAMattributes NH HI NM MD AS XS \
   --outSAMunmapped Within \
   --outSAMheaderHD @HD VN:1.4 SO:unsorted \
   --outFilterMultimapNmax 20 \
   --outFilterScoreMinOverLread 0.33 \
   --outFilterIntronMotifs RemoveNoncanonicalUnannotated \
   --alignIntronMax 200000 \
   --alignMatesGapMax 200000 \
   --alignSJDBoverhangMin 1 \
   --quantMode TranscriptomeSAM \
   --readFilesCommand zcat \
   --outSAMheaderCommentFile ${OUT_COMMENT_FILE} \
   --outSAMattrRGline ${CUSTOM_RG_LINE} \
   --outFileNamePrefix ${OUTPUT_DIR} \
   --runThreadN ${THREADS} \
   --genomeDir ${REFERENCE_DIR} \
   --sjdbGTFfile ${TRANSCRIPTOME_GTF_FILE} \
   --readFilesIn {MATCHED_PAIR_FIRST_MATE_FILE} {MATCHED_PAIR_SECOND_MATE_FILE}

• Sort BAM by coordinates using bamsort (biobambam2 version 2.0.87)

   bamsort \
   fixmate=1 \
   inputformat=bam \
   level=1 \
   inputthreads=${THREADS} \
   outputthreads=${THREADS} \
   I=${STAR_ALIGNED_OUT_BAM} \
   tmpfile=${TMP_FILE} \
   O=${OUT_SORTED_BAM}

• Mark duplicates using bammarkduplicates2 (biobambam2 version 2.0.87)

   bammarkduplicates2 \
   md5=1 \
   index=1 \
   tmpfile=${TEMP_FILE} \
   markthreads=${THREADS} \
   md5filename=${OUT_BAM_MD5} \
   indexfilename=${OUT_BAM_INDEX} \
   M=${OUT_MET} \
   I=${SORTED_BAM} \
   O=${OUT_DUP_MARKED_BAM}

• Sort BAM by read names using bio (biobambam2 version 2.0.87) so that HTSeq-count can use

   bamcollate2 \
   collate=1 \
   inputformat=bam \
   outputformat=bam \
   level=1 \
   exclude=SECONDARY,SUPPLEMENTARY \
   filename=${OUT_DUP_MARKED_BAM} \
   O=${NAME_SORTED_BAM}

• HTSeq-count (Version 0.7.2)

   htseq-count \
   --format=bam \
   --order=name \
   --stranded="no" \
   --type="exon" \
   --idattr="gene_id" \
   --mode="union" \
   --quiet \
   ${NAME_SORTED_BAM} ${HTSEQ_GTF}