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cannot read data from simulated FASTQ files

Hi All,

after some suffering I realized, that perhaps my simulated paired-end fastq files are not correct in some way. (I found in Bowtie2 log 'no input read files were valid' and Trimmomatic couldn't handle these files either)

So, below there is a part of one of my paired end sequences, I don't know what's wrong with that:

@s_forw_0_1-17:0-81195210_1-5957-4/1
TCCCTCAGTTTCCCCATCTGTGAAATGGGCTGGCCATGCTTAACCCCTGGAGTTGCCAAGGTAGCCCATCAGGGAACACAGCGCCCCTGTACCTCAGGCACTCCC
+
??AA??B?DDD?D.DBGGCCCFIEIEHIIHIIIIIEIIHFHIGHIIHIHHIIIIIHIFFHIIHHHGHHIIHHHIIHHHFDI-IIHHHHIHCHHHIDIFHI,*HHH
@s_forw_0_1-17:0-81195210_1-5957-2/1
CCCTCAGTTTCCCCATCTGTGAAATGGGCTGGCCATGCTTAACCCCTGGAGTTGCCAAGGTAGCCCATCAGGGAACACAGCGCCCCTGTACCTCAGGCACTCCCT
+
?A???BBBDDADBDDBGGGGGGIIIFIHFHIIFHIHIDCHIFEIHIIGHIEGHDFGH+IHGIHHEC@5FEHIHHHIHH?IGFFIGHDHHIFIHHFHHGHIHCHHH
@s_revc_0_2-17:0-81195210_1-5957-4/1
GGGCGGGCTCAGGAAGAGGAGGCCAAGGACGGCCAAGAGAGGGGTGGGAGCAGGTGAGGACTGAGTGACCGTCCCGTCCAACAGGTGGACAAGACCGAAGACAAG
+
?????B?BBBDDEDDDFGGEGGHCHIHBHEHIIIHHIHHIIIHIHIHIHGIHEHHHHIHI==IIIFHHIIHHIAFAAIIHDIIGDHIHFHHGHFFHHIDFHHHGH
@s_revc_0_2-17:0-81195210_1-5957-2/1
GGTCACTCAGTCCTCACCTGCTCCCACCCCTCTCTTGGCCGTCCTTGGCCTCCTCTTCCTGAGCCCGCCCATCCGGCTGCTGCAGCCGGGCCTGGTCACGGTCCC
+
??AAABBBDDDDBDDEGGFCGFFIHFHIIIHHHFGGDFIICHHFIFFHDHHIIIHHIIIHHFIIHEEHHAFHHIHFIIH=GEIFIFHIIHHHIHHEHHHHHHFDH
@s_forw_1_1-17:0-81195210_1-5957-4/1
CAGGTGAGGACTGAGTGACCGTCCCGTCCAACAGGTGGACAAGACCGAAGACAAGTCCCTGGAGGAGCGGGGCCGCATCCTCATCTCGCTCAAGTACAGCTCACA
+
?,A??BBBBDDDBD@DFFGCFFIIIFIIFIIIIHIHHHIHHHHHFHIHIIIA=HIIIIIIHHICDHHHIHIFIHIEIHIH=HIBIH8HFIHHHGHHIG=HHI=HF
@s_forw_1_1-17:0-81195210_1-5957-2/1
CAGCTCACAGAAGCAAGGCCTGCTGGTAGGCATCGTGCGGTGCGCCCACCTGGCCGCCATGGACGCCAACGGCTACTCGGACCCCTACGTAAAAACGTGAGTGTG
+
??9??B?@DDDDDD6EGFGGGCHIB@ICICIIIIHHFIIIDHHIAHIIIHIHHFGIFIIIBHIIFHHHHHDHIHFIDIIIHHHCIII5HH+IAIHGHFHIHHGHH
@s_forw_1_2-17:0-81195210_1-5957-4/1
ACGGCACACTCACGTTTTCACGTAGGGGTCCGAGTAGCCGTTGGCGTCCATGGCGGCCAGGTGGGCGCACCGCACGATGCCTACCAGCAGGCCTTGCTTCTGTGA
+
?,????B?BBD-DDBDGGFGGCIAIHH/IFHHGAEIFIDHIIHHHIIHIF?HGIHIFIIFFHIHHIHIHHDHGHIIFH?IICGCII>EI.HIHCHIIHHHFH4HF
@s_forw_1_2-17:0-81195210_1-5957-2/1
GGTGCGCCCACCTGGCCGCCATGGACGCCAACGGCTACTCGGACCCCTACGTGAAAACGTGAGTGTGCCGTGCGCGTGACCACCTGCCACGTCTTCACCTCCAAG
+
????ABBBDDBDDDDDFCGGGFHHIIFHHIHIC=H9IEHHIIHGHIHICHHIHIFHIHIEHFIHFHHEHIHGHIHIICHIH@IAIIHIHFIIHIFHHHI=HHGDG
@s_forw_0_1-17:0-81195210_1-11156-4/1
CCCGATGAACTCATTGTGCCGGAATTTGTCCTCGTCACACACAGAGATCCTAGAGGGGGCGGTGGTGAGGGGCACAGCCAGTGCCTCAGACGCACTGGGCATGGT
+
?????B@BD@DDDDDDCGECFGIIFIFHIAIIHFEGIHFHHHFGIIHIIICCHIIIHIHFIEFHHHIGIIIFHIHHIDHHIHHHHFH#EIFDIHHFIGHHGFHHB
@s_forw_0_1-17:0-81195210_1-11156-2/1
CCTTCCTCACCTCCACCCCCTTGACTCTCCATGCTCACCTCCCCGGTCTCCCCTCCCCTCTCACTCTGCCCCTCATGAGTCCCATCACAGGCAGGAAGTTATGCC
+

the Trimmomatic command I used:

java -jar /opt/Trimmomatic-0.33/

trimmomatic-0.33.jar PE -trimlog ./fastq/Trimmomatic/trimlog_control_0_D1.txt ./fastq/control_0_D4_1.fq ./fastq/control_0_D4_2.fq ./fastq/Trimmomatic/Paired/control_0_D1_forward-paired.fastq ./fastq/Trimmomatic/Unpaired/control_0_D1_forward-unpaired.fastq ./fastq/Trimmomatic/Paired/control_0_D1_reverse-paired.fastq ./fastq/Trimmomatic/Unpaired/control_0_D1_reverse-unpaired.fastq ILLUMINACLIP:/opt/Trimmomatic-0.33/adapters/TruSeq3-PE.fa:2:40:10

and the output of it:

TrimmomaticPE: Started with arguments: -trimlog ./fastq/Trimmomatic/trimlog_control_0_D1.txt ./fastq/control_0_D4_1.fq ./fastq/control_0_D4_2.fq ./fastq/Trimmomatic/Paired/control_0_D1_forward-paired.fastq ./fastq/Trimmomatic/Unpaired/control_0_D1_forward-unpaired.fastq ./fastq/Trimmomatic/Paired/control_0_D1_reverse-paired.fastq ./fastq/Trimmomatic/Unpaired/control_0_D1_reverse-unpaired.fastq ILLUMINACLIP:/opt/Trimmomatic-0.33/adapters/TruSeq3-PE.fa:2:40:10
Multiple cores found: Using 4 threads
Using PrefixPair: 'TACACTCTTTCCCTACACGACGCTCTTCCGATCT' and 'GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT'
ILLUMINACLIP: Using 1 prefix pairs, 0 forward/reverse sequences, 0 forward only sequences, 0 reverse only sequences
Quality encoding detected as phred33
Input Read Pairs: 204352 Both Surviving: 204352 (100.00%) Forward Only Surviving: 0 (0.00%) Reverse Only Surviving: 0 (0.00%) Dropped: 0 (0.00%)
TrimmomaticPE: Completed successfully

Thank you for any help!

fastq fileformat
• 2,075 views • link
updated by Ram Ram • written by anon • anon

I suspect the problem relates to something else. I can't see anything wrong here.

• 0 views • link
written by scchess scchess

It could be a newline or path issue, or something else. Writing out the full command for Trimmomatic and/or bowtie may help to resolve the issue.

• 0 views • link
written by SES SES

I'm sure that the path is correct.

• 0 views • link
written by anon • anon

2 answers

Have you tried this: http://genome.sph.umich.edu/wiki/FastQValidator ? Its pretty old, but might work.

If you didn't spot the bug in the first few lines, you're not going to see it manually - I would definitely look for a validator, or write some tests into your code to make sure its doing what you think it should be doing :)

• 0 views • link
updated by Ram Ram • written by John John 
@HWI-EAS209_0006_FC706VJ:5:58:5894:21141#ATCACG/1
TTAATTGGTAAATAAATCTCCTAATAGCTTAGATNTTACCTTNNNNNNNNNNTAGTTTCTTGAGATTTGTTGGGGGAGACATTTTTGTGATTGCCTTGAT
+HWI-EAS209_0006_FC706VJ:5:58:5894:21141#ATCACG/1
efcfffffcfeefffcffffffddf`feed]`]_Ba_^__[YBBBBBBBBBBRTT\]][]dddd`ddd^dddadd^BBBBBBBBBBBBBBBBBBBBBBBB

What you can see above is a nice an useful FastQ data taken from http://en.wikipedia.org/wiki/FASTQ_format

@HWI-EAS209_0006_FC706VJ ->You can see the name of the machine, then

5 -> the flowcell line

58: the "parcel" within the flowcell line (tesela)

5894:21141: the coordinates within that tesela that contains the DNA you are sequencing

#ATCACG is optional and is present only when using a barcode

/1 if paired...

I miss this into your data.

@s_forw_1_2-17:0-81195210_1-5957-4/1 
ACGGCACACTCACGTTTTCACGTAGGGGTCCGAGTAGCCGTTGGCGTCCATGGCGGCCAGGTGGGCGCACCGCACGATGCCTACCAGCAGGCCTTGCTTCTGTGA
+
?,????B?BBD-DDBDGGFGGCIAIHH/IFHHGAEIFIDHIIHHHIIHIF?HGIHIFIIFFHIHHIHIHHDHGHIIFH?IICGCII>EI.HIHCHIIHHHFH4HF
• 0 views • link
updated by Ram Ram • written by Antonio R. Franco Antonio R. Franco

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