Sequence read and quality do not match - Trimmomatic

Question

[csjlee08]

Hi, I am working with GBS data. I have demultiplexed the reads and am trying to trim the adapters using Trimmomatic. The error I get is:

java.lang.RuntimeException: Sequence and quality length don't match: 'TGCAGCCCAAAACAAAAACTCGAATTGAAAAGTGGGAGTTTGAGGCGAAGAAAGAGACCGAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGATCTCGTATGCCGTCTTCTGCTTGAAAAAAAAAAAAATGAAAAAAAAAAAAAAAACACGAAAAAAAAAAAAACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAAATAAAAAAAAAAAAAAAAAATAAAAAAAATATAATAACAAAAAAATAAATAAATAATATAAAAGTGAACATGATAAGAACTAA' vs '499:3:000000000-BFRBP:1:1101:14660:2815 1:N:0:1'

I am not sure how to fix this as it appears the length is very different. Any insight is appreciated. I will put some examples of the demultiplexed fasta below.

Christine

   greg@Genomics:/media/greg/StorageD/gbs3/GBSpipeline$ ./GBS_pipeline.pl trim_reads /media/greg/StorageD/gbs3/GBSpipeline/trimmomatic-master/classes/trimmomatic.jar illuminaadaptersFR.fa
given is experimental at ./GBS_pipeline.pl line 114.
when is experimental at ./GBS_pipeline.pl line 117.
when is experimental at ./GBS_pipeline.pl line 161.
when is experimental at ./GBS_pipeline.pl line 196.
when is experimental at ./GBS_pipeline.pl line 232.
Calling trim_reads ...
ERROR:  indexshortcolfull.txt does not exist or is unreadable.
greg@Genomics:/media/greg/StorageD/gbs3/GBSpipeline$ ./GBS_pipeline.pl GBS_function2.pl
given is experimental at ./GBS_pipeline.pl line 114.
when is experimental at ./GBS_pipeline.pl line 117.
when is experimental at ./GBS_pipeline.pl line 161.
when is experimental at ./GBS_pipeline.pl line 196.
when is experimental at ./GBS_pipeline.pl line 232.
Calling GBS_function2.pl ...
ERROR: Trimmomatic did not complete successfully:e: BC01       
'java -classpath /media/greg/StorageD/gbs3/GBSpipeline/trimmomatic-master/classes/trimmomatic.jar org.usadellab.trimmomatic.TrimmomaticPE -threads 1 -phred33 -trimlog logs/BC01.trim.log /media/greg/StorageD/gbs3/GBSpipeline/processedreads/demultiplex/BC01_Adzuki_R1.fastq /media/greg/StorageD/gbs3/GBSpipeline/processedreads/demultiplex/BC01_Adzuki_R2.fastq /media/greg/StorageD/gbs3/GBSpipeline/processedreads/trim/BC01_Adzuki_R1-p.fastq /media/greg/StorageD/gbs3/GBSpipeline/processedreads/trim/BC01_Adzuki_R1-s.fastq /media/greg/StorageD/gbs3/GBSpipeline/processedreads/trim/BC01_Adzuki_R2-p.fastq /media/greg/StorageD/gbs3/GBSpipeline/processedreads/trim/BC01_Adzuki_R2-s.fastq ILLUMINACLIP:illuminaadaptersFR.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36 ' exited with value 1
TrimmomaticPE: Started with arguments:  -threads 1  -phred33  -trimlog  logs/BC01.trim.log  /media/greg/StorageD/gbs3/GBSpipeline/processedreads/demultiplex/BC01_Adzuki_R1.fastq  /media/greg/StorageD/gbs3/GBSpipeline/processedreads/demultiplex/BC01_Adzuki_R2.fastq  /media/greg/StorageD/gbs3/GBSpipeline/processedreads/trim/BC01_Adzuki_R1-p.fastq  /media/greg/StorageD/gbs3/GBSpipeline/processedreads/trim/BC01_Adzuki_R1-s.fastq  /media/greg/StorageD/gbs3/GBSpipeline/processedreads/trim/BC01_Adzuki_R2-p.fastq  /media/greg/StorageD/gbs3/GBSpipeline/processedreads/trim/BC01_Adzuki_R2-s.fastq  ILLUMINACLIP:illuminaadaptersFR.fa:2:30:10  LEADING:3  TRAILING:3  SLIDINGWINDOW:4:15  MINLEN:36 
 Using Long Clipping Sequence: 'AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT'
 Using Long Clipping Sequence: 'CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAA' 
 ILLUMINACLIP: Using 0 prefix pairs, 2 forward/reverse sequences, 0 forward only sequences, 0 reverse only sequences
 Exception in thread "main"  java.lang.RuntimeException: Sequence and quality length don't match: 'TGCAGCCCAAAACAAAAACTCGAATTGAAAAGTGGGAGTTTGAGGCGAAGAAAGAGACCGAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGATCTCGTATGCCGTCTTCTGCTTGAAAAAAAAAAAAATGAAAAAAAAAAAAAAAACACGAAAAAAAAAAAAACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAAATAAAAAAAAAAAAAAAAAATAAAAAAAATATAATAACAAAAAAATAAATAAATAATATAAAAGTGAACATGATAAGAACTAA' vs '499:3:000000000-BFRBP:1:1101:14660:2815 1:N:0:1'



    @M05499:3:000000000-BFRBP:1:1101:17626:1036 1:N:0:1
TGCAGAATAGGAAACTATGTCAGGAAGGAAACCTAGCTGAACCATGCTGTCAAGAAAATCGCATGCTTCTACAGCCCGAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGATCTCGTATGCCGTCTTCTGCTTGAAAAAAAAAAAAAAAAAACAAAAAAAAAACAAAACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATACATAAAAAAAAAAAATAAAAAAAAAAATATATAAATATATAATAATATATGACGTCTATATTAAAGATTGT
+
CGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG@CGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG@555>54*4:,<*=<*<**4=<=,2==FFGGGGGCEGE2::::?/2:*2C2:2/::*2:EGG>GC*:*21:C1:*3+303<2<AE*:*:CGF<3<<@*:C:5C22233++<+3<C0<+39CF?97<+3+**1;;79+++3+00+++0
@M05499:3:000000000-BFRBP:1:1101:21721:1044 1:N:0:1
TGCAGAGGAAATTAAGTGAAGTACTCCAGACACAAAGTATGGAAAAGTGTCTATAACTTCCCCCCCAGGACCTACTCCCCAACCTAGAGTAGCTAGGTGGGGAAGCAAAATTAAACCTTGTTCATACATGGGCTTCTCGGTTACGAAATGAGCCACTTCAAAAAGGTTCATTGCTCCGAGAACGGAAGAGCGGTTCAGCAGGAATGCCGAAACCGATATCGTATGCCGTCTTCTGCTTGAAAAAAAAAAAAAAAAAATAAAAAAAAAAAATAAAAACACAAGACAGAATGAATAGAG
+
CGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGEGGGGGGGGF+BFFEFGGG?F?FEFGGGGFFGGGECEFGEGF,CBFFF@3@+:888C3@:,555@3777765>F3777>51=464444*24+452,24**2*,<,,,<74?FGGGGGGGCEG*:CE/*<<@@:*:1CE8EG++3+0*****/**0*2<3392+0+0+
@M05499:3:000000000-BFRBP:1:1101:10857:1069 1:N:0:1
TGCAGAGAAGATTCAAGAAACCCAAATCAAAACCCTTGCCACGACGTCACCGTAAAATCAATTCCGCCGCTACAACACTCACTGCGCAATGAAAAGGGAGGACGCCCCCTAGCCACCGAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGATCTCGTATGCCGTCTTCTGCTTGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATAATATAACATAAAAAATATATACAAACAATAATATCCTAACAATAAATA
+
CGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFFFGGGGGGGGGGGGGGGG<>FGFGF:>FFG7CFFEFGGFFF8CFFGGGGGGF:::EGEG::CEEEGGGGGEGGGG:EEEEG::CC>EGGGGGGGCEE*?EGGGCG:C:*0A:+3+3<33+3<++3?*+33+<9E9+***+3++++3++++*00+3++0+

[lieven.sterck]

did you check your files? It appears to me that it's not taken the quality line with that sequence but rather the ID of the next read?

Can you check that specific line (with grep for instance) with some 'context' lines before and after? or count the numbers of lines in the file and check if that's a multiple of 4?

[h.mon]

It seems you are using this pipeline: GBSpipeline. What is the content of the config file? What is the "indexshortcolfull.txt" file?