trimmomatic for quality trimming

Question

[blooming.daisy333]

I am using trimmomatic for quality trimming of Paired reads. however, i am unable to get equal numbrer of reads in paired read files. Can anyone please help me out to resolve this issue. the command I am using is as follows:

java -jar trimmomatic-0.36.jar -phred33 PE /path/to/dir/SRR9590_1.fastq /path/to/dir/SRR9590_2.fastq SRR9590_1P.fq SRR9590_1U.fq SRR9590_2P.fq SRR9590_2U.fq ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36

[GenoMax]

I suggest you give bbduk.sh from BBMap suite a try.

[Carlo Yague]

How many reads do you have in each file ? SRR9590_1P.fq and SRR9590_2P.fq should have the same number of reads. How many reads are dropped ? can you show us trimmomatic output ?

[blooming.daisy333]

I am newbie to NGS analysis. I am consistently getting the same error. the number of reads in 1P file is 362080 while in 2P is 361087 in current session. Sometimes I even get the different number (I mean not this one)

[blooming.daisy333]

here is the output for new trial:

TrimmomaticPE: Started with arguments:

 /data/sata4/memona/SRR959590_1.fastq /data/sata4/memona/SRR959590_2.fastq SRR959590_g_1P.fq SRR959590_g_1U.fq SRR959590_g_2P.fq SRR959590_g_2U.fq ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36
java.io.FileNotFoundException: /data/sata4/memona/Trimmomatic-0.36/TruSeq3-PE.fa (No such file or directory)
        at java.io.FileInputStream.open(Native Method)
        at java.io.FileInputStream.<init>(FileInputStream.java:146)
        at org.usadellab.trimmomatic.fasta.FastaParser.parse(FastaParser.java:54)
        at org.usadellab.trimmomatic.trim.IlluminaClippingTrimmer.loadSequences(IlluminaClippingTrimmer.java:110)
        at org.usadellab.trimmomatic.trim.IlluminaClippingTrimmer.makeIlluminaClippingTrimmer(IlluminaClippingTrimmer.java:71)
        at org.usadellab.trimmomatic.trim.TrimmerFactory.makeTrimmer(TrimmerFactory.java:32)
        at org.usadellab.trimmomatic.Trimmomatic.createTrimmers(Trimmomatic.java:59)
        at org.usadellab.trimmomatic.TrimmomaticPE.run(TrimmomaticPE.java:536)
        at org.usadellab.trimmomatic.Trimmomatic.main(Trimmomatic.java:80)
Quality encoding detected as phred33
Exception in thread "main" java.lang.RuntimeException: Sequence and quality length don't match: 'AGGAGCTGGGAAGCAAAGGAGGTCAGACAAGAAAGGAGCAGATAGGGACTGAAGGGTACCAGGAAATGGGGCGAAAAGGTGGACTGAGTACGATGGAC' vs '@?=DDBDD?DHHFBFHJAC3ADHCHGIAF>B3CGEC;DHIIAG����**�:��ZY8�`�
                    u��Wi��%^|\�/V�Œ���@'
        at org.usadellab.trimmomatic.fastq.FastqRecord.<init>(FastqRecord.java:25)
        at org.usadellab.trimmomatic.fastq.FastqParser.parseOne(FastqParser.java:89)
        at org.usadellab.trimmomatic.fastq.FastqParser.next(FastqParser.java:179)
        at org.usadellab.trimmomatic.TrimmomaticPE.processSingleThreaded(TrimmomaticPE.java:63)
        at org.usadellab.trimmomatic.TrimmomaticPE.process(TrimmomaticPE.java:311)
        at org.usadellab.trimmomatic.TrimmomaticPE.run(TrimmomaticPE.java:539)
        at org.usadellab.trimmomatic.Trimmomatic.main(Trimmomatic.java:80)

the number of reads in 1P file is 362080 while in 2P is 361087 in current session. Sometimes I even get the different number. kindly help me to sort out this issue. thanks

Answer 1

[finswimmer]

Hello,

first let's check if there are the same number of line in both fastq files. So what's the output of wc -l /path/to/dir/SRR9590_1.fastq and wc -l /path/to/dir/SRR9590_2.fastq?

If it's the same number we can go on. In your output above you have this message:

java.io.FileNotFoundException: /data/sata4/memona/Trimmomatic-0.36/TruSeq3-PE.fa

Check if this file realy doesn't exist. If there is a similar named file adopt your command line to this. I have found some threads where the authors guess that the error thread "main" java.lang.RuntimeException: Sequence and quality length don't match: arise due to the missing adaptor file.

fin swimmer

[Carlo Yague]

Yes this is probably the cause of your issue.

The error means that the reference file for adapter trimming is missing or not at the expected location. Locate this file (TruSeq3-PE.fa) then replace ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 by ILLUMINACLIP:correct/path/to/TruSeq3-PE.fa:2:30:10.

[blooming.daisy333]

thank you so much . it has worked correctly. Now i am getting ewual number of reads. thanks again