Trimmomatic Unpaired files

[daanum]

Hi,

I run the following command in Trimmomatic:
java -jar /home/devgen/bin/trimmomatic-0.36.jar PE -phred33 C95VLANXX-2046D-01-01-01_L003_R1.fastq C95VLANXX-2046D-01-01-01_L003_R2.fastq N6_F_P.fastq N6_F_U.fastq N6_R_P.fastq N6_R_U.fastq ILLUMINACLIP:adapters/TruSeq3-PE.fa:2:30:10 MINLEN:25 AVGQUAL:20 -trimlog N6L3.log

Am I running it correctly?

I have RNASeq Paired end data from HiSEq2500.

The output looks like this:

TrimmomaticPE: Started with arguments:
-phred33 C95VLANXX-2046D-01-01-01_L003_R1.fastq C95VLANXX-2046D-01-01-01_L003_R2.fastq N6_F_P.fastq N6_F_U.fastq N6_R_P.fastq N6_R_U.fastq ILLUMINACLIP:adapters/TruSeq3-PE.fa:2:30:10 MINLEN:25 AVGQUAL:20 -trimlog N6L3.log
Using PrefixPair: 'TACACTCTTTCCCTACACGACGCTCTTCCGATCT' and 'GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT'
ILLUMINACLIP: Using 1 prefix pairs, 0 forward/reverse sequences, 0 forward only sequences, 0 reverse only sequences
Input Read Pairs: 16228554 Both Surviving: 12432585 (76.61%) Forward Only Surviving: 3793730 (23.38%) Reverse Only Surviving: 1926 (0.01%) Dropped: 313 (0.00%)
TrimmomaticPE: Completed successfully

---------
Is it normal to get only 76% surviving in both reads? What do I do with the upaired ones? Unpaired forward file has 23% of reads which is quiet a lot. Should I just leave this file and only use paired-survived file for alignment with tophat2?


Any advise will be greatly appreciated.

Thanks.

[daanum]

Any suggestions on this will be greatly appreciated.

[Brian Bushnell]

Well... if I recall correctly, the default behavior of Trimmomatic when adapters are discovered is to drop one of the reads, because it will simply be the reverse-complement of its mate. I think it is possible to disable this behavior, which would make things simpler as you would then have only paired reads as output. The default behavior of BBDuk when adapter-trimming is to trim and output both reads in the pair, so that you don't end up with singletons unnecessarily.

You CAN use both the paired and singleton reads for alignment - and generally, you should, unless you already have plenty of coverage - but it's even better, in my opinion, to simply not discard mates in the first place when you don't have to.

[daanum]

Thanks. I did not understand this when you say trimmomatic keeps paired reads when option keepbothreads is set to 'true'.could you please graphically explain what it does? I am new to rnaseq so please excuse my silly questions.
Thanks

[jackfrost]

Thanks. I did not understand this when you say trimmomatic keeps paired reads when option keepbothreads is set to 'true'.could you please graphically explain what it does? I am new to rnaseq so please excuse my silly questions.
Thanks

They do a pretty good job in the manual (TrimmomaticManual_V0.32.pdf) with explaining each step including graphics which is often helpful. Its probably a good idea to become familiar with each step so you can use the program most appropriately based on the specifics of your data and your experiment. From my experience this is time always well spent when using a new bioinformatics tool. Good luck.