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How do you decide to set the run to SE or PE on trimmomatic?

How do you decide to set the run to SE or PE on trimmomatic?

next-gen rna-seq
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updated by Ram Ram • written by sam • sam

I ran this command and it was successful in trimmomatic but there is no trimmomatic-logs folder in the results. What am I missing?

java -jar /path/to/trimmomatic-0.39.jar \
  PE -trimlog trimlog_20191205.B-2744XJ_3.txt 20191205.B-2744XJ_3_1.fastq.gz 20191205.B-2744XJ_3_2.fastq.gz \
  SRR2589044_1.trim.fastq.gz SRR2589044_1un.trim.fastq.gz \
  SRR2589044_2.trim.fastq.gz SRR2589044_2un.trim.fastq.gz \
  SLIDINGWINDOW:4:20 MINLEN:25 ILLUMINACLIP:TruSeq3-PE.fa:2:40:15

TrimmomaticPE: Started with arguments:
  -trimlog trimlog_20191205.B-2744XJ_3.txt 20191205.B-2744XJ_3_1.fastq.gz 20191205.B-2744XJ_3_2.fastq.gz SRR2589044_1.trim.fastq.gz SRR2589044_1un.trim.fastq.gz SRR2589044_2.trim.fastq.gz SRR2589044_2un.trim.fastq.gz SLIDINGWINDOW:4:20 MINLEN:25 ILLUMINACLIP:TruSeq3-PE.fa:2:40:15
Multiple cores found: Using 4 threads
Using PrefixPair: 'TACACTCTTTCCCTACACGACGCTCTTCCGATCT' and 'GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT'
ILLUMINACLIP: Using 1 prefix pairs, 0 forward/reverse sequences, 0 forward only sequences, 0 reverse only sequences
Quality encoding detected as phred33
Input Read Pairs: 266245409 Both Surviving: 222729178 (83.66%) Forward Only Surviving: 37659701 (14.14%) Reverse Only Surviving: 3517537 (1.32%) Dropped: 2338993 (0.88%) 
TrimmomaticPE: Completed successfully
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updated by Ram Ram • written by sam • sam

Please do not post output of commands as answers. Answers are meant to be NEW answers for the original question at the top of the post.

It is tempting to ask for help for every small thing but there is a learning process that needs to happen. Bioinformatics is not just running commands but it is also about understanding what the program does with its options. Experiment and try things out. When you have specific questions or run into issues ask. We are here to help.

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written by GenoMax GenoMax

1 answer

If you have two FASTQ files per sample (e.g., something_R1.fastq and something_R2.fastq), then you have paired end (PE) reads. If you have only one FASTQ file per sample (e.g., something_R1.fastq only), then you have single end (SE) reads.

You call Trimmmomatic on whatever data you have (SE or PE). There really isn't much of a choice here unless you're going to discard one of the read pairs from a PE sequencing run.

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written by Dunois • Dunois

Thank you!!

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written by sam • sam

Do you know how to decide between phred33 and phred64?

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written by sam • sam

Most current data should be phred33 format. Old (6+ years) data may be in phred64 format. You can use testformat.sh from BBMap suite if you are not sure.

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written by GenoMax GenoMax

Thank you for your response!!! Do you have any idea on how to decide which parameter to change for more quality results while trimming with Trimmomatic?

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written by sam • sam

That will change with every dataset. If you have checked FastQC report then that will give you some guidance. For starters you can simply use the default recommended by trimmomatic manual. They should generally be sufficient.

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written by GenoMax GenoMax

Honestly, just use fastp instead. Should be much easier to use.

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written by Dunois • Dunois

Use the default settings which are optimized unless you have a good reason to change them, which is almost never the case.

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written by ATpoint ATpoint

There really isn't much of a choice here unless you're going to discard one of the read pairs from a PE sequencing run.

If one has PE data then the files should be trimmed together to keep reads in sync post-trimming.

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written by GenoMax GenoMax

I decided on PE. I mean in general. Out of these parameters.

PE [-version] [-threads <threads>] [-phred33|-phred64] [-trimlog
<trimLogFile>] [-quiet] [-validatePairs] [-basein <inputBase> |
<inputFile1> <inputFile2>] [-baseout <outputBase> | <outputFile1P>
<outputFile1U> <outputFile2P> <outputFile2U>] <trimmer1>...

We are having trouble trimming the adapters properly, so what would you suggest which parameter I can change to experiment?

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updated by Ram Ram • written by sam • sam

We are having trouble trimming the adapters properly

What made you decide that is the case? You should generally know from the kit used to prepare the libraries what kind of adapters your data has.

Your data does not NEED to have adapters present. Only if the length of sequencing is longer than the insert size then the read will have read-through into adapter at 3'-end. Having no adapter sequence is a good sign.

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written by GenoMax GenoMax

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