Put technical sequences into separate files

Separating based on location

Let’s say we have barcodes, UMIs, and the biological sequence at specified locations. For example, 10X (version 3) chemistry has the following format:

Barcode (16 bp): File 0 (R1.fastq.gz), position 0 to 16
UMI (12 bp): File 0 (R1.fastq.gz), position 16-28
cDNA: File 1 (R2.fastq.gz), entire read

We can extract into separate files as follows:

splitcode -x "0:0<barcode>0:16,0:16<umi>0:28,1:0<cdna>1:-1" --x-only --nFastqs=2 --gzip R1.fastq.gz R2.fastq.gz

Three files will be generated: barcode.fastq.gz, umi.fastq.gz, cdna.fastq.gz

Note

Note that quality scores will NOT be preserved (all quality scores will be replaced with K).

Also note that in lieu of a config.txt file, we supplied the extraction pattern on the command line using -x (although we could use a config file with just one line containing the extraction string preceded by @extract).

Separating based on tag identification

Let’s revisit the example, where, for R1.fastq, we have a 5-bp Barcode A, followed by a variable length region (region 1), followed by a 5-bp/6-bp barcode B, followed by an 8-bp UMI 3-bp’s after barcode B, followed by a variable length region (region 2) that procedes until the end of the read. For R2.fastq, the entire sequence is the cDNA sequence (except the last 4 bp’s are trimmed).

We want to extract the barcodes, the UMI, region 1+2, and the cDNA into separate files.

Here, we’ll create the following config.txt file:

@extract <barcode_A{{@grp_A}}>,<barcode_B{{@grp_B}}>,{{grp_B}}3<umi[8]>,{{grp_A}}<region_1>{{grp_B}},{{grp_B}}3<region_2>0:-1,1:0<cdna>1:-1
@trim-3 0,4
groups ids             tags      distances   next      maxFindsG locations
grp_A  Barcode_A1      AAGGA     1           {{grp_B}} 1         0:0:5
grp_A  Barcode_A2      GTGTG     1           {{grp_B}} 1         0:0:5
grp_A  Barcode_A3      CGTAT     1           {{grp_B}} 1         0:0:5
grp_B  Barcode_B1      GCGCAA    0           -         1         0:5:100
grp_B  Barcode_B2      CCCGT     0           -         1         0:5:100

We can extract into separate files as follows:

splitcode -c config.txt --x-only --nFastqs=2 --gzip R1.fastq R2.fastq

Six files will be generated:

barcode_A.fastq.gz
barcode_B.fastq.gz
umi.fastq.gz
region_1.fastq.gz
region_2.fastq.gz
cdna.fastq.gz