[nareshvasani]

Thanka a bunch.

You can use cutadapt to trim both 5' and 3' bps. The fastx_clipper can only trim 3' end. When you use cutadapt, you must use cutadapt -g firstly, and use the processed sequence to do cutadapt -a. If you use -g and -a at the same time, it will only cut one end.

[nareshvasani]

I did the same way.

Thanks for feedback.

I always use the fastx_trimmer; you can use the -f and -l options to set the first and the last base to be kept.

[Oyster_lab]

So, just following up on this topic. It has been incredibly helpful. We shouldn't trim the first bases at the 5' end and try to perform the de novo assembly that way correct?

Thanks!

[Brian Bushnell]

It depends on the library prep. Illumina fragment libraries typically have adapters on the right (3') end, so if you trimmed to the left from the adapter you'd lose all of your genomic sequence. For long mate pair libraries, the answer depends on the protocol.

[Oyster_lab]

Thanks for your reply, Brian.
I have mRNA Illumina 100bp paired end reads. I have already removed the adapters, but still have that same the high variation on GC% at the 5' end. For the library prep, TruSeq mRNA prep was used, that's why I am guessing I have the same 5' end bias described before on my dataset. Any thoughts?

[Brian Bushnell]

BBDuk can trim a set number of bases on the left or right side of a read. However, there are some library-prep protocols that are biased, especially near the read start, and thus have suspicious base-frequency histograms, even though they are correct. So, before you trim, I suggest you map the reads to a reference (even the lowest-quality assembly is OK) to determine whether there is actually a higher error rate in the first X bases of the read. If not, then you should not trim them.

With an assembly, you can determine it like this:

bbmap.sh in=reads.fq mhist=mhist.txt qhist=qhist.txt

This will give you histograms of the average qualities by read position, and match/substitution/insertion/deletion/N rates by read position. That will allow you to determine whether the stated read quality is accurate, and thus whether you need to trim the ends of reads.

If you want to trim a set number of bases on each side, you can use BBDuk's "ftl" (force-trim left) and "ftr" (force-trim right) flags to set the limits of where to trim.

[Brian Bushnell]

The fragmentation sites may be biased, depending on how fragmentation was done. Try mapping with BBMap and using the 'mhist' output, which shows the error rate by read position. If the error rate on the 5' end is not much higher than anywhere else, there's no need to trim it.

[arkanion]

Use Trimmomatic

[liguanghao]

I use TrimGalore to trim adapter and the fastqc result also show that there are biases in 5' end, several kmers occurring.