I am aligning a fastq file as follows:
cutadapt -j 8 --quality-base 33 -l 20 \
-o - /home/user/Documents/analyses/CRISPR-screens/screen3/D16.fq.gz |
~/bowtie2-2.4.3-linux-x86_64/bowtie2 --no-hd -p 8 \
-t -N 0 -x ~/moffat_tko3 - |
sed '/XS:/d' | cut -f3 | sort | uniq -c > ~/D16.guidecounts.txt
The expected output is similar to this:
head D16.guidecounts.txt
329 A1BG_sgA1BG_1
669 A1BG_sgA1BG_2
65 A1BG_sgA1BG_3
397 A1BG_sgA1BG_4
100 A1CF_sgA1CF_1
731 A1CF_sgA1CF_2
396 A1CF_sgA1CF_3
536 A1CF_sgA1CF_4
427 A2ML1_sgA2ML1_1
978 A2ML1_sgA2ML1_2
However, there is no output from this command as bowtie2
gets stuck. cutadapt
works fine, but when that step is done, nothing happens for hours and it should only take a few minutes.
I have tried to take a subset of reads using head
and tail
as follows:
zcat D16.fq.gz | head -10000 | gzip -c > D16head.fq.gz
zcat D16.fq.gz | tail -10000 | gzip -c > D16tail.fq.gz
What confuses me is that these files don't freeze bowtie2
and align well (> 90% alignment rates). Other files from the same sequencing run also align fine (NovaSeq SP flow cell 50 bp paired-end).
As far as I can tell the original fastq file looks fine:
zcat D16.fq.gz | head -12
@A00489:1088:HKN2TDRXY:2:2101:1334:1172 1:N:0:GCCAAT
GCCCAGTGACCCTTTCACGGGTTTTAGAGCTAGAAATAGCAAGTTAAAAT
+
FFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,,FFFF
@A00489:1088:HKN2TDRXY:2:2101:1606:1172 1:N:0:GCCAAT
AAAGTTCTATCTGTTGGGCGGTTTTAGAGCTAGAAATAGCAAGTTCAAGT
+
FFFFFFFFFFFFFFFFFFFFF:FFFFFFF:FFFFF:FFFFFFFFF,FFFF
@A00489:1088:HKN2TDRXY:2:2101:1624:1172 1:N:0:GCCAAT
GCGACGGTTCATCTCCGCAAGTTTTAGAGCTAGAAATAGCAAGTTAAAAT
+
FFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFF:FFFFFFFF,FFFF
zcat D16.fq.gz | tail -12
@A00489:1088:HKN2TDRXY:2:2278:30644:37059 1:N:0:GCCAAT
AGGTCATCCACTCTAGACCTGTTTTAGAGCTAGAAATAGCAAGTTAAAAT
+
FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF
@A00489:1088:HKN2TDRXY:2:2278:30915:37059 1:N:0:GCCAAT
ATGAGGAGGGTGATGTTCCGGAGTTTTAGAGCTAGAAATAGCAAGTTAAA
+
FF:FFFFFFFFFFFFFFFFFFFFFFF,FFFFFF:FFFFFFFFFFFFFFFF
@A00489:1088:HKN2TDRXY:2:2278:31638:37059 1:N:0:GCCAAT
GAGTGTGGTTGAGATCCCAAGTTTTAGGCTAGAAATAGCAAGTTAAAATA
+
FF:FF:F:FFFFFFFF,FFFFFF:FF::FFFFFFFFFFFFFFFFFFFFFF
Does anyone know what could be wrong?