#umi_tools whitelist --stdin data/12-22-3_L2_1.fq.gz \

#                    --bc-pattern="(?P<discard_1>CTCTTTCCCT)(?P<cell_1>.{10})(?P<discard_2>ACGACGCTCTTCGAGTGATTGCTTGTGACGCCTT)(?P<cell_2>.{8})(?P<umi_1>.{12})T{4}.*" \

#                    --set-cell-number=2000 \

#                    --extract-method=regex \

#                    --log2stderr > whitelist.txt;

#

## Step 3: Extract barcdoes and UMIs and add to read names

#umi_tools extract --bc-pattern="(?P<discard_1>CTCTTTCCCT)(?P<cell_1>.{10})(?P<discard_2>ACGACGCTCTTCGAGTGATTGCTTGTGACGCCTT)(?P<cell_2>.{8})(?P<umi_1>.{12})T{4}.*" \

#                  --extract-method=regex \

#                  --stdin data/12-22-3_L2_1.fq.gz \

#                  --stdout data/12-22-3_L2_1_extracted.fastq.gz \

#                  --read2-in data/12-22-3_L2_2.fq.gz \

#                  --read2-out=data/12-22-3_L2_2_extracted.fastq.gz \

#                  --whitelist=whitelist.txt;

# Step 4: Map reads

STAR --runThreadN 4 \

     --genomeDir /share/nas5/huangls/test/scRNA-seq.1/ref/refdata-gex-mm10-2020-A/star/ \

     --readFilesIn data/12-22-3_L2_2_extracted.fastq.gz \

     --readFilesCommand zcat \

     --outFilterMultimapNmax 1 \

     --outSAMtype BAM SortedByCoordinate;

# Step 5: Assign reads to genes

/share/work/biosoft/subread/latest/bin/featureCounts \

               -a /share/nas5/huangls/test/scRNA-seq.1/ref/refdata-gex-mm10-2020-A/genes/genes.gtf \

              -o gene_assigned \

              -R BAM Aligned.sortedByCoord.out.bam \

              -T 4;

samtools sort Aligned.sortedByCoord.out.bam.featureCounts.bam -o assigned_sorted.bam;

samtools index assigned_sorted.bam;

# Step 6: Count UMIs per gene per cell

umi_tools count --per-gene --gene-tag=XT \

   --assigned-status-tag=XS --per-cell \

   --wide-format-cell-counts \

   -I assigned_sorted.bam -S counts.tsv.gz