Question: Output files are empty after adapter trimming using trim galore
0
gravatar for nalandaatmi
3.7 years ago by
nalandaatmi60
United States
nalandaatmi60 wrote:

Dear All,

I used trim galore for adapter trimming.

Command : trim_galore --paired -a GAGAGCGATCCTTGC -a2 AGATCGGAAGAGC 2002_AGCTAGTG_L002_R1.all.fastq.gz 2002_AGCTAGTG_L002_R2.all.fastq.gz -o ./ --path_to_cutadapt $HOME/.local/bin/cutadapt

Output files:

2004_GCGTATCA_L002_R1.all_val_1.fq  and 2004_GCGTATCA_L002_R2.all_val_2.fq (Both files are empty). 

This is the summary of trim galore

=== Summary ===

Input filename: /Sample_2002/2002_AGCTAGTG_L002_R1.all.fastq.gz

Total reads processed:               6,289,520
Reads with adapters:                 2,963,516 (47.1%)
Reads written (passing filters):     6,289,520 (100.0%)

Total basepairs processed:   635,241,520 bp
Quality-trimmed:              12,351,960 bp (1.9%)
Total written (filtered):    568,191,752 bp (89.4%)

=== Adapter 1 ===

Sequence: GAGAGCGATCCTTGC; Type: regular 3'; Length: 15; Trimmed: 2963516 times.

=== Summary ===

Input filename: //Sample_2002/2002_AGCTAGTG_L002_R2.all.fastq.gz

Total reads processed:               6,289,520
Reads with adapters:                 1,832,060 (29.1%)
Reads written (passing filters):     6,289,520 (100.0%)

Total basepairs processed:    37,737,120 bp
Quality-trimmed:                 580,324 bp (1.5%)
Total written (filtered):     34,632,200 bp (91.8%)

=== Adapter 1 ===

Sequence: AGATCGGAAGAGC; Type: regular 3'; Length: 13; Trimmed: 1832060 times.

Last 3 lines of my trim galore log file:

Total number of sequences analysed: 6289520

Number of sequence pairs removed because at least one read was shorter than the length cutoff (20 bp): 6289520 (100.00%)

Deleting both intermediate output files 2002_AGCTAGTG_L002_R1.all_trimmed.fq.gz and 2002_AGCTAGTG_L002_R2.all_trimmed.fq.gz

ADD COMMENTlink modified 3.7 years ago by h.mon27k • written 3.7 years ago by nalandaatmi60

It appears that one of the reads after trimming is failing the default length cutoff (20 bp) so this results in effective removal of all data. Did the FastQC analysis indicate a problem with adapters?

ADD REPLYlink written 3.7 years ago by genomax70k

Hi,

Yeah, the reads are contaminated with adapter sequence.

ADD REPLYlink written 3.7 years ago by nalandaatmi60

If one of the two reads is affected you may be able to salvage one good read by trimming the files independently. Not sure if it helps you though.

ADD REPLYlink written 3.7 years ago by genomax70k

Hi nalandaatmi, Which kind of platform adapter 'GAGAGCGATCCTTGC' have you used in trim galore. I checked for Illumina,Nextera and few others but was unable to search.

ADD REPLYlink written 3.5 years ago by vaibhav.vsk0
0
gravatar for h.mon
3.7 years ago by
h.mon27k
Brazil
h.mon27k wrote:

TrimGalore! default minimal length is 20bp, you may use --length to alter it, but unless you are sequencing something you expect to be really short, your data has problems. Is this microRNA sequencing?

ADD COMMENTlink written 3.7 years ago by h.mon27k

Hi, human Mitochondrial DNA has been sequenced.

This is what I noticed in trim galore log file:

No quality encoding type selected. Assuming that the data provided uses Sanger encoded Phred scores (default)

Path to Cutadapt set as: '/data2/test/.local/bin/cutadapt' (user defined)
1.9
Cutadapt seems to be working fine (tested command '/data2/test/.local/bin/cutadapt --version')
Writing report to './2002_GCGTATCA_L002_R1.all.fq_trimming_report.txt'

I didn't mention the Phred33 or Phred64 in my trim galore command because I am just interested in trimming the adapters not based on the quality. Will that have impacted my analysis?

 

ADD REPLYlink modified 3.7 years ago • written 3.7 years ago by nalandaatmi60
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