scPreproc

1) Demultiplexing (01_divmux.codon):

• Have codon and seq installed.

• Run script like this:

codon run -plugin seq -release -D BC_LEN=16 -D BC_START=29 -D MIN_READS_IN_BC=200 01_divmux.codon <Path_I1_gzipped_or_not> <Path_R1_unzipped> <Path_R2_unzipped> <Path_whitelist> <Path_output_stats.tsv> <Path_reads_per_barcode_stats.tsv> <sam_header.txt> <Hamming_distance_allowed>

e.g.:

codon run -plugin seq -release -D BC_LEN=16 -D BC_START=29 -D MIN_READS_IN_BC=200 01_divmux.codon ScKDMA_S1_R2_001.fastq.gz ScKDMA_S1_R1_001.fastq ScKDMA_S1_R3_001.fastq whitelist_10xATAC_737K-cratac-v1.txt ScKDMA_S1_BC_stats.tsv ScKDMA_S1_reads_per_barcode.tsv sam_header.txt 1

Values for BC_LEN, BC_START and MIN_READS_IN_BC need to be passed when invoking codon!

• Output: <Path_R1_unzipped>_correct.fastq <Path_R3_unzipped>_correct.fastq <Path_output_stats.tsv> <Path_reads_per_barcode_stats.tsv> <sam_header.txt>

2) Alignment (02_divalign.sh):

• Uses bwa, or faster yet, bwa-mem2 binaries, samtools and GATK.

• Run script like this:

./02_divalign.sh <exp_type> <modality> <sample_name> <Path_R1_correct> <Path_R2_correct> <threads> <path_bwa> <path_bwarefDB> <PathGATK> <RemDups> <PathSamtools> <PathOutputBam> <PathOutputPicardDupStats> <sam_header> <min_good_reads_in_cells>

e.g.:

./02_divalign.sh nanoCNT modA ScKDMA_S1 R1_correct.fq R2_correct.fq 8 /home/bwa-mem2-2.2.1_x64-linux/bwa-mem2 /home/refBWAmem2/hg19.fa /home/gatk-4.5.0.0/gatk true /home/micromamba/envs/ali/bin/samtools /home/testing/TEST.bam /home/testing/TEST_DupMetrics.txt /home/testing/sam_header.txt 200

• Output: <PathOutputBam> <PathOutputBam>.bai <PathOutputPicardDupStats> In script directory: <sample_name>-<modality>-<exp_type>_ProperPairedMapped_reads_per_barcode.tsv