Question

Trimmomatic PE - output , low Surviving / Forward Only

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24 months ago

01de ▴ 10

Hi

I'm a student making my first attempt at analysis some data. They were processed on NextSeq 150 bp paired end. The quality of my reads appears to be pretty good looking at it with fasctqc/multiqc , although the adapter content does seem to be high.

MultiQC - Pre-trimming quality

MultiQC - Adapter Content

I've my adaptor removal using "ILLUMINACLIP:TruSeq3-PE.fa:2:30:10:2" which seemed to be a pretty common setting for Trimmomatic. Looking at surviving read output I've got from trimmomatic, am i right in thinking these are rather low ?

Input Read Pairs: 21749931 Both Surviving: 17321416 (79.64%) Forward Only Surviving: 4428493 (20.36%) Reverse Only Surviving: 0 (0.00%) Dropped: 22 (0.00%)
Input Read Pairs: 29312959 Both Surviving: 14225497 (48.53%) Forward Only Surviving: 15087439 (51.47%) Reverse Only Surviving: 0 (0.00%) Dropped: 23 (0.00%)
Input Read Pairs: 25108389 Both Surviving: 9625828 (38.34%) Forward Only Surviving: 15482512 (61.66%) Reverse Only Surviving: 0 (0.00%) Dropped: 49 (0.00%)
Input Read Pairs: 28341291 Both Surviving: 13160173 (46.43%) Forward Only Surviving: 15181099 (53.57%) Reverse Only Surviving: 0 (0.00%) Dropped: 19 (0.00%)
Input Read Pairs: 26062118 Both Surviving: 17567347 (67.41%) Forward Only Surviving: 8494753 (32.59%) Reverse Only Surviving: 0 (0.00%) Dropped: 18 (0.00%)
Input Read Pairs: 21518085 Both Surviving: 8773336 (40.77%) Forward Only Surviving: 12744722 (59.23%) Reverse Only Surviving: 0 (0.00%) Dropped: 27 (0.00%)
Input Read Pairs: 24399016 Both Surviving: 11339058 (46.47%) Forward Only Surviving: 13059923 (53.53%) Reverse Only Surviving: 0 (0.00%) Dropped: 35 (0.00%)
Input Read Pairs: 26929633 Both Surviving: 18386746 (68.28%) Forward Only Surviving: 8542855 (31.72%) Reverse Only Surviving: 0 (0.00%) Dropped: 32 (0.00%)
Input Read Pairs: 38208049 Both Surviving: 19638876 (51.40%) Forward Only Surviving: 18569149 (48.60%) Reverse Only Surviving: 0 (0.00%) Dropped: 24 (0.00%)
Input Read Pairs: 23513417 Both Surviving: 17493860 (74.40%) Forward Only Surviving: 6019544 (25.60%) Reverse Only Surviving: 0 (0.00%) Dropped: 13 (0.00%)
Input Read Pairs: 32211396 Both Surviving: 12670482 (39.34%) Forward Only Surviving: 19540886 (60.66%) Reverse Only Surviving: 0 (0.00%) Dropped: 28 (0.00%)
Input Read Pairs: 23504113 Both Surviving: 18896861 (80.40%) Forward Only Surviving: 4607236 (19.60%) Reverse Only Surviving: 0 (0.00%) Dropped: 16 (0.00%)
Input Read Pairs: 26559578 Both Surviving: 8626098 (32.48%) Forward Only Surviving: 17933458 (67.52%) Reverse Only Surviving: 0 (0.00%) Dropped: 22 (0.00%)
Input Read Pairs: 27088465 Both Surviving: 17346145 (64.04%) Forward Only Surviving: 9742304 (35.96%) Reverse Only Surviving: 0 (0.00%) Dropped: 16 (0.00%)
Input Read Pairs: 23560546 Both Surviving: 13269766 (56.32%) Forward Only Surviving: 10290768 (43.68%) Reverse Only Surviving: 0 (0.00%) Dropped: 12 (0.00%)
Input Read Pairs: 23215260 Both Surviving: 17096697 (73.64%) Forward Only Surviving: 6118528 (26.36%) Reverse Only Surviving: 0 (0.00%) Dropped: 35 (0.00%)
Input Read Pairs: 28340841 Both Surviving: 8470755 (29.89%) Forward Only Surviving: 19870009 (70.11%) Reverse Only Surviving: 0 (0.00%) Dropped: 77 (0.00%)
Input Read Pairs: 33104009 Both Surviving: 12043911 (36.38%) Forward Only Surviving: 21060051 (63.62%) Reverse Only Surviving: 0 (0.00%) Dropped: 47 (0.00%)
Input Read Pairs: 25039143 Both Surviving: 16616396 (66.36%) Forward Only Surviving: 8422743 (33.64%) Reverse Only Surviving: 0 (0.00%) Dropped: 4 (0.00%)
Input Read Pairs: 22756628 Both Surviving: 17214428 (75.65%) Forward Only Surviving: 5542185 (24.35%) Reverse Only Surviving: 0 (0.00%) Dropped: 15 (0.00%)
Input Read Pairs: 40291036 Both Surviving: 33270962 (82.58%) Forward Only Surviving: 7020066 (17.42%) Reverse Only Surviving: 0 (0.00%) Dropped: 8 (0.00%)

nextgen trimmomatic • 1.3k views

ADD COMMENT • link 24 months ago by 01de ▴ 10

score 2 · Accepted Answer · 2022-05-09

you have a considerable amount of adapter read through and thus should expect a relatively high amount of read loss. In this case you may want to optimize the ILLUMINACLIP parameters accordingly.

A less well known ILLUMINACLIP parameter is keepBothReads. This is by default set to 'false' and thus why you are not getting any reverse reads returned:

ILLUMINACLIP:<fastaWithAdaptersEtc>:<seed mismatches>:<palindrome clip
threshold>:<simple clip threshold>:<minAdapterLength>:<keepBothReads>

keepBothReads: After read-though has been detected by palindrome mode, and the adapter sequence removed, the reverse read contains the same sequence information as the forward read, albeit in reverse complement. For this reason, the default behaviour is to entirely drop the reverse read. By specifying 'true' for this parameter, the reverse read will also be retained, which may be useful e.g. if the downstream tools cannot handle a combination of paired and unpaired reads.