Question: Length of reads in a RNAseq experiment
0
gravatar for Carlos Caicedo
2.8 years ago by
Colombia/Universidad de Antioquia
Carlos Caicedo130 wrote:

Should all the reads of my experiment be of the same length if I am going to perform a de novo assembly?

The raw data contained reads of the same length but after the trimming process by quality some of them are reduced. I would like to know if there are some effect in the downstream analysis with this reads. (The majority of the reads remain of the same length and only a few reads have been trimmed)

Thanks

Carlos Andrés.

rna-seq • 671 views
ADD COMMENTlink modified 2.8 years ago by igor7.7k • written 2.8 years ago by Carlos Caicedo130

Generally i've heard that you don't want to trim your RNA-seq reads as it could mess with downstream analysis (of course you'd remove adapter contamination).

ADD REPLYlink written 2.8 years ago by Sinji2.8k

It would be god to know what kind of trimming you did, like the program and command used.

ADD REPLYlink written 2.8 years ago by geek_y9.7k

I used Trimmomatic The “paired-end data” option was selected and the trimming steps were:

ILLUMINACLIP which cut adapter and other illumina-specific sequences from the read. The parameter for this step were:

  • Truseq3 (Paired-ended, For miSeq and Hiseq)
  • Maximum mismatch count which will still allow a full match to be performed: 2
  • How accurate the match between the two 'adapter ligated' reads must be for PE palindrome read alignment: 30
  • How accurate the match between any adapter etc. sequence must be against a read: 10

SLIDINGWINDOW which perform a sliding window trimming, cutting once the average quality within the window falls below a threshold. The parameter for this step were:

  • Number of bases to average across: 4
  • Average quality required: 20
ADD REPLYlink modified 2.8 years ago • written 2.8 years ago by Carlos Caicedo130
2
gravatar for igor
2.8 years ago by
igor7.7k
United States
igor7.7k wrote:

You should trim your reads before assembly. That's a perfectly reasonable assumption. I think any assembler will accept variable-length trimmed reads.

ADD COMMENTlink modified 2.8 years ago • written 2.8 years ago by igor7.7k
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