fastq headers problem with Trinity

Question

[agata]

I downloaded data as SRA file and used fastq_dump according to Trinity recommendations.

fastq-dump --skip-technical  --readids --read-filter pass --dumpbase --defline-seq '@$sn[_$rn]/$ri' --split-files ./SRR.sra

Then I run quality control with FastQC and trimmed out adapters with trimmomatic.

My headers looks like this:

head -n 8 SRR5874687.1_pass_1_trim.fastq 
@/1
GACCGTAGCCGTGGTATTTACTTCACTCAAGACTGGTGTTCAATGCCAGGTGTTATGCCAGTTGCTTCAGGTGGTATTCACGTATGGCACATGCCAGCTT
+SRR5874687.1.171.1 length=100
?1BDDD8B:AC@DEA:ACHAAFH?+2?1??FD9CFH9BDGDBBDGECBDFCFHG>F=@GFFHGIGIH@AHEEHF4;@C3.>BA3AAD=5;,:>C@>><CC
@/1
CTTTTACTGAATCCATGGGGTGTTTCTTATTCTTAGCTCAAAGTCTGTACATGTTGTGCACGTGCTGAAACCGCGTGTGCCGGTTGCGCGAGTCCTCTCA
+SRR5874687.1.172.1 length=100
?@@FFADDHFFFFGECGIGI@GHIJ?HHDGH?FH?D@GGHGGGGGIIGCGHDEHIHIICFFHHICGGCDHECBBBCBBDDDDD=B>?B>B5953:>:@>:

head -n 8 SRR5874687.1_pass_2_trim.fastq 
@/2
CACCGAACTGAAGACATGCGTCATCACCGAAGATTTCAACTAAAGCTGGCATGTGCCATACGTGAATACCACCTGAAGCAACTGGCATAACACCTGGCAT
+SRR5874687.1.171.2 length=100
@@@DFFDDHBFHDHGBFG@@C<@F>??CFHIH0??FFIGII<BBC@FCFCHGH.7777=D;AHEFB@?7;;>BEC;@CCCC??ACBCCCCCCC?CC@?CC
@/2
CTGGACAACGCGCCGCAATATTGCAGCTTATTAGTTTGGTGATGAGAGGACTCGCGCAACCGGCACACGCGGTTTCAGCACGTGCACAACATGTACAGAC
+SRR5874687.1.172.2 length=100
?@@FBDDDFHDHHJJJIGHIIJJGGHIGI?FH<DFHJJJCF@GHFHGHIGHHEEEDDDDDDDDDDDDDD@BBBBDDEDDDDDBDDDDDDDDDDDEEEECB

At this point Trinity had problem with empty kmer25.

Primarily I was thinking that the problem is with header position (3rd line instead of 1st), so I asked here for help. First I moved headers from third line to the first with awk proposed method and then used bbmap to add slashes (/1 and /2).

Now, headers look like this:

head -n 8 slashed_biostar_1.fastq 
@SRR5874687.1.171.1 length=100 /1
GACCGTAGCCGTGGTATTTACTTCACTCAAGACTGGTGTTCAATGCCAGGTGTTATGCCAGTTGCTTCAGGTGGTATTCACGTATGGCACATGCCAGCTT
+
?1BDDD8B:AC@DEA:ACHAAFH?+2?1??FD9CFH9BDGDBBDGECBDFCFHG>F=@GFFHGIGIH@AHEEHF4;@C3.>BA3AAD=5;,:>C@>><CC
@SRR5874687.1.172.1 length=100 /1
CTTTTACTGAATCCATGGGGTGTTTCTTATTCTTAGCTCAAAGTCTGTACATGTTGTGCACGTGCTGAAACCGCGTGTGCCGGTTGCGCGAGTCCTCTCA
+
?@@FFADDHFFFFGECGIGI@GHIJ?HHDGH?FH?D@GGHGGGGGIIGCGHDEHIHIICFFHHICGGCDHECBBBCBBDDDDD=B>?B>B5953:>:@>:

head -n 8 slashed_biostar_2.fastq 
@SRR5874687.1.171.2 length=100 /2
CACCGAACTGAAGACATGCGTCATCACCGAAGATTTCAACTAAAGCTGGCATGTGCCATACGTGAATACCACCTGAAGCAACTGGCATAACACCTGGCAT
+
@@@DFFDDHBFHDHGBFG@@C<@F>??CFHIH0??FFIGII<BBC@FCFCHGH.7777=D;AHEFB@?7;;>BEC;@CCCC??ACBCCCCCCC?CC@?CC
@SRR5874687.1.172.2 length=100 /2
CTGGACAACGCGCCGCAATATTGCAGCTTATTAGTTTGGTGATGAGAGGACTCGCGCAACCGGCACACGCGGTTTCAGCACGTGCACAACATGTACAGAC
+
?@@FBDDDFHDHHJJJIGHIIJJGGHIGI?FH<DFHJJJCF@GHFHGHIGHHEEEDDDDDDDDDDDDDD@BBBBDDEDDDDDBDDDDDDDDDDDEEEECB

But this time Trinity not recognizing read name formatting: [SRR5874687.1.171.2]

I have one guess, that maybe if put number of read after a space at the end it could help, like: [SRR5874687.1.171.2 1]. Somebody knows how to do it automatically?

Do you know what messed up headers? Because they didn't change after using trimmomatic (they were exactly the same as after using fastq-dump on raw data (that worked perfectly till now) ).

Answer 1

[agata]

After all, the format of the header that enables me to run Trinity is as follow:

@1/1
GACCGTAGCCGTGGTATTTACTTCACTCAAGACTGGTGTTCAATGCCAGGTGTTATGCCAGTTGCTTCAGGTGGTATTCACGTATGGCACATGCCAGCTT
+SRR5874687.1.171.1 1 length=100
?1BDDD8B:AC@DEA:ACHAAFH?+2?1??FD9CFH9BDGDBBDGECBDFCFHG>F=@GFFHGIGIH@AHEEHF4;@C3.>BA3AAD=5;,:>C@>><CC
@2/1
CTTTTACTGAATCCATGGGGTGTTTCTTATTCTTAGCTCAAAGTCTTACATGTTGTGCACGTGCTGAAACCGCGTGTGCCGGTTGCGCGAGTCCTCTCA
+SRR5874687.1.172.1 2 length=100
?@@FFADDHFFFFGECGIGI@GHIJ?HHDGH?FH?D@GGHGGGGGIIGCGHDEHIHIICFFHHICGGCDHECBBBCBBDDDDD=B>?B>B5953:>:@>:
@3/1
TCACATTATATTTCTGTTTTTGATCAACAATATCGTTTACCACGTAATCGTTATCTAAAACGCAACCCATTAAAAACATTGAAAAAAACGACTTTATTAG
+SRR5874687.1.173.1 3 length=100
B@@FFDFDHHHHHIIJJJJJJJHIDJJJJIHIIHHJIJGG<**??F8?08??/?)8=FH@=@'B/AH=(7?CEEEA9(;;;@CCD?D<<9505?C3>@3:

So in the first line after "@" has to be the number of a read and the unified code "/1" or "/2" that respcets first or second read of paired reads. In the third line after "+" should be kept the original ID of the read, and after a space the number of read should be repeated. After the next space may be present the information about length of the read (not necessary).

I used "awk" command to edit headers - here code for first read of pair:

Edit the first line: awk '/^@\/1/{sub(/\/1/,++i"/1")}1' SRR5874687.1_pass_1_trim.fastq > out.txt and then the third line: awk '/^+SRR/{sub(/ /, " " ++i " ")}1' out1.txt > SRR5874687.1_pass_1_trim_awk.fastq