Split Multimer Sequences at motif in FASTQ

Question

[schmau]

Hi,

I have FASTQ file containing PCR multimeres and i need to split the sequences with a known primer sequence and kind of demultiplex them, but i want my primer to remain:

My input looks like this (primer bold)

@A00877:568:HVV57DSXY:4:1101:27724:1408 1:N:0

GGGTCAGTAGCGGAC GGGAACTGCATCACGCAATACGACTCACTATA GGGTCAGTAGCGGAC......

+

FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF.......

output:

@A00877:568:HVV57DSXY:4:1101:27724:1408 1:N:0:1

GGGTCAGTAGCGGAC GGGAACTGCATCACGCAATACGACTCACTATA

+

FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF

@A00877:568:HVV57DSXY:4:1101:27724:1408 1:N:0:2

GGGTCAGTAGCGGAC......

+

FFFFFFFFFFFFFFFFFFFFFF.......

Thanks a lot!

[cpad0112]

with cutadapt:

input:

$ cat test.fq                                                                                                                                                                        

@A00877:568:HVV57DSXY:4:1101:27724:1408 1:N:0
GGGTCAGTAGCGGACGGGAACTGCATCACGCAATACGACTCACTATAGGGTCAGTAGCGGAC
+
FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF
@A00877:568:HVV57DSXY:4:1102:27724:1408 1:N:0
GGGTCAGTAGCGGACGGGAAACGTCGCACGCAATACGACTCACTATAGGGTCAGTAGCGGAC
+
FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF
@A00877:568:HVV57DSXY:4:1103:27724:1408 1:N:0
GGGTCAGTAGCGGACGGGAACTGCACGTCAGCTGGCGACTCACTATAGGGTCAGTAGCGGAT
+
FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF

output:

$ cutadapt -e 0 -g GGGTCAGTAGCGGAC...GGGTCAGTAGCGGAC  --action=retain --discard-untrimmed --quiet test.fq                                                                            

@A00877:568:HVV57DSXY:4:1101:27724:1408 1:N:0
GGGTCAGTAGCGGACGGGAACTGCATCACGCAATACGACTCACTATAGGGTCAGTAGCGGAC
+
FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF
@A00877:568:HVV57DSXY:4:1102:27724:1408 1:N:0
GGGTCAGTAGCGGACGGGAAACGTCGCACGCAATACGACTCACTATAGGGTCAGTAGCGGAC
+
FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF

updated to remove primer at the end:

$ cutadapt -e 0  -g GGGTCAGTAGCGGAC...GGGTCAGTAGCGGAC  --action=retain --discard-untrimmed  --quiet test.fq | cutadapt -u -17 - --quiet

@A00877:568:HVV57DSXY:4:1101:27724:1408 1:N:0
GGGTCAGTAGCGGACGGGAACTGCATCACGCAATACGACTCACTA
+
FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF
@A00877:568:HVV57DSXY:4:1102:27724:1408 1:N:0
GGGTCAGTAGCGGACGGGAAACGTCGCACGCAATACGACTCACTA
+
FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF

-e 0 allowed errors zero as the third read differs by one base at 3' end.

Answer 1

[GenoMax]

If I understand what you want then following should work. From BBMap suite.

$ more test.fq
@A00877:568:HVV57DSXY:4:1101:27724:1408 1:N:0
GGGTCAGTAGCGGACGGGAACTGCATCACGCAATACGACTCACTATAGGGTCAGTAGCGGAC
+
FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF
@A00877:568:HVV57DSXY:4:1102:27724:1408 1:N:0
GGGTCAGTAGCGGACGGGAAACGTCGCACGCAATACGACTCACTATAGGGTCAGTAGCGGAC
+
FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF
@A00877:568:HVV57DSXY:4:1103:27724:1408 1:N:0
GGGTCAGTAGCGGACGGGAACTGCACGTCAGCTGGCGACTCACTATAGGGTCAGTAGCGGAC
+
FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF

Do the trimming.

$ bbduk.sh -Xmx2g in=test.fq outu=stdout.fq literal=GGGTCAGTAGCGGAC ktrim=r restrictright=20 -da k=7

Version 38.35

0.020 seconds.
Initial:
Memory: max=2147m, total=2147m, free=2129m, used=18m

Added 9 kmers; time:    0.005 seconds.
Memory: max=2147m, total=2147m, free=2126m, used=21m

Input is being processed as unpaired
Started output streams: 0.012 seconds.
@A00877:568:HVV57DSXY:4:1101:27724:1408 1:N:0
GGGTCAGTAGCGGACGGGAACTGCATCACGCAATACGACTCACTATA
+
FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF
@A00877:568:HVV57DSXY:4:1102:27724:1408 1:N:0
GGGTCAGTAGCGGACGGGAAACGTCGCACGCAATACGACTCACTATA
+
FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF
@A00877:568:HVV57DSXY:4:1103:27724:1408 1:N:0
GGGTCAGTAGCGGACGGGAACTGCACGTCAGCTGGCGACTCACTATA
+
FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF
Processing time:        0.005 seconds.

Input:                      3 reads         186 bases.
KTrimmed:                   3 reads (100.00%)   45 bases (24.19%)
Total Removed:              0 reads (0.00%)     45 bases (24.19%)
Result:                     3 reads (100.00%)   141 bases (75.81%)

Time:                           0.025 seconds.
Reads Processed:           3    0.12k reads/sec
Bases Processed:         186    0.01m bases/sec

Change stdout.fq to a file name to write the result out to a file instead of STDOUT. Use in1= in2= outu1= outu2= if you have paired-end data.