Hi,
I am attempting to demultiplex barcoded 100bp paired-end illumina short-read sequencing data with cutadapt following these instructions: https://cutadapt.readthedocs.io/en/stable/guide.html#demultiplexing
I only want to retain read pairs where the barcodes were found and trimmed/removed in both reads of a pair.
However cutadapt is outputting untrimmed second reads despite having specified the "--untrimmed-paired-output" argument.
Full details of my analysis are as follows (cutadapt 1.17 with Python 3.6.5):
Command-line parameters:
cutadapt -g file:demultiplex_barcode-file_1.fasta -G file:demultiplex_barcode-file_1.fasta -e 0.25 --no-indels --untrimmed-output Input_1.fastq.gz.demultiplex.unknown.fastq --untrimmed-paired-output Input_2.fastq.gz.demultiplex.unknown.fastq -o {name}1.fastq -p {name}2.fastq Input_1.fastq.gz Input_2.fastq.gz
demultiplex_barcode-file_1.fasta:
>Input_Rep1_read
^GCCGAATT
>Input_Rep2_read
^CGGCAATT
>Input_Rep3_read
^GAACGTTC
Before cutadapt demultiplexing (example read1 FASTQ entry in Input_1.fastq.gz):
@D3FCO8P1:231:C49LBACXX:7:1101:2911:2101 1:N:0:
GCCGAATTTGCAGTTTGAACAAAGCAAGAACTTACCCCAAACAATTAGTGGAATTGGCAAAAGAAGAAGACAAAGCCACCCCAAGTTAGATTTCGATCCT
+
CCCFFFFFHHHHHJJJJJJJJJJJJJIJJIIJJJIJJJJJIIIIIJIJGGIIIIJGIGGGIJIIJCGHEHEC@D@CA@C>BDDDDCDCCCCDCDCBD?@>
Before cutadapt demultiplexing (example read2 FASTQ entry in Input_2.fastq.gz):
@D3FCO8P1:231:C49LBACXX:7:1101:2911:2101 2:N:0:
CCGAATTAAAATGTCCAATGTTCCAACCTACAGGATCGAAATCTAACTTGGGGTGGCTTTGTCTTCTTCTTTTGCCAATTCCACTAATTGTTTGGGGTAA
+
CCCFFFFFHHHHHJJJJJJIJJJJJJIJJJIJJJJJJJJIJJJJGHGIJIIJJIIDBE@GGHFFFHDHFFCFFEA6>;@ACCACC;;>>:>CCA@BBDB3
After cutadapt demultiplexing (example read1 FASTQ entry in Input_Rep1_read1.fastq):
@D3FCO8P1:231:C49LBACXX:7:1101:2911:2101 1:N:0:
TGCAGTTTGAACAAAGCAAGAACTTACCCCAAACAATTAGTGGAATTGGCAAAAGAAGAAGACAAAGCCACCCCAAGTTAGATTTCGATCCT
+
HHHHHJJJJJJJJJJJJJIJJIIJJJIJJJJJIIIIIJIJGGIIIIJGIGGGIJIIJCGHEHEC@D@CA@C>BDDDDCDCCCCDCDCBD?@>
After cutadapt demultiplexing (example read2 FASTQ entry in Input_Rep1_read2.fastq):
@D3FCO8P1:231:C49LBACXX:7:1101:2911:2101 2:N:0:
CCGAATTAAAATGTCCAATGTTCCAACCTACAGGATCGAAATCTAACTTGGGGTGGCTTTGTCTTCTTCTTTTGCCAATTCCACTAATTGTTTGGGGTAA
+
CCCFFFFFHHHHHJJJJJJIJJJJJJIJJJIJJJJJJJJIJJJJGHGIJIIJJIIDBE@GGHFFFHDHFFCFFEA6>;@ACCACC;;>>:>CCA@BBDB3
As you can see, the barcode was not matched in the second read but both reads nevertheless still appear in the supposedly trimmed output files (Input_Rep1_read1.fastq and Input_Rep1_read2.fastq).
I have tried specifying "--pair-filter=any" although this is the default setting. Neither specifying "any" nor "both" makes any difference to this read pair being retained despite the second read being untrimmed.
Any help would be appreciated!
Thanks,
Andre
