Output files are empty after adapter trimming using trim galore

Question

[nalandaatmi]

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

[GenoMax]

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?

[nalandaatmi]

Hi,

Yeah, the reads are contaminated with adapter sequence.

[GenoMax]

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.

[vaibhav.vsk]

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.

Answer 1

[h.mon]

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?

[nalandaatmi]

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?