PCR duplicata and de novo assembly ??

Question

[Picasa]

Hi,

Sorry I am a beginner in NGS field I used SGA preqc to assess the quality of my data.

This program estimates the PCR duplicate proportion to 20%.

1) First, I am not sure to understant what is PCR duplicate, what is the difference with the sequencing coverage ?

2) 20%: Is it a lot ?

3) If yes, how to remove them ? is it important prior to a de novo assembly ?

Answer 1

[Medhat]

There is two types of duplication PCR duplication and optical duplication, we remove duplicates mainly to reduce recurrent errors.

• PCR duplication are introduced during library preparation

  you can find nice info about it here also

• optical duplicates (Illumina) are obtained when a single cluster of reads is part of two adjacent tiles' on the same slide and used to compute two read calls separately

here you can use this tool to remove them
Note: while using the tool I suggested

To remove the duplicate records from the resulting file, set the REMOVE_DUPLICATES parameter to true

[Picasa]

Thanks for your support.

1) Did you have any good experience with removing those duplicates ? (your N50 improved a lot )

2) Should I remove it on raw reads or trimmed reads ?

[Medhat]

• from the word duplication leaving them will not change N50 "cause actually they are the same no information will be introduced by leaving them" on the other hand it will introduce bias in downstream analysis.
• first you trimme your reads then remove them

Answer 2

[Satyajeet Khare]

20% duplicates are not a lot. And I think for assembly they should not matter much. These are duplicate reads in your sample. For quantitative analyses such as ChIP-seq and RNA-Seq, they can pose a big problem.