Trimmomatic : Why is output different? ; filtering with row -> q20 -> q30 vs row -> q30

Question

[SSK]

Hi.

I did quality control with condition >q30 by using Trimmomatic.

At first, I filtered my reads (already filtered with q>20), and the rate of both surviving was about 30%.

However, when I filtered row reads with >q30, the both surviving rate was about 98%.

Why is this?

For your information, when I filtered row reads with >q20, the both surviving rate was about 98%.

Thanks.

[SSK]

I tried to re-analysis with condition below

1. raw reads ---filter at q20 ---> -----filter at q30 ---> filtered reads
2. raw reads ---filter at q30 ---> filtered reads

Then, I got exactly same rate of both surviving.

In fact, I found my code was bad.

Sorry, Carambakaracho, arup and everyone.

[Carambakaracho]

Your numbers are confusing to me. You retain 98% of the read pairs (both surviving) when filtering with either >q20 and >q30? When exactly do you keep only 30%? When you filter at q20?

From the title I guess it's

100% - filter at q20 - retain 30% pairs - subsequent filter at q30 - retain 98% pairs

100% - filter at q30 - retain 98% pairs

[SSK]

Sorry, Carambakaracho.

I intended that it was

100% - filter at q20 - retain 98% pairs - subsequent filter at q30 - retain 30% pairs

100% - filter at q30 - retain 98% pairs

[Arup Ghosh]

Have you checked the raw read quality statistics with FastQC or similar tools?

[SSK]

Yes, I checked my reads quality. But, I can't find something wrong with my reads. Almost all my reads data was high quality(>q30).

[Arup Ghosh]

Can you share the statistics here?