removing of duplication in RNA seq

Question: removing of duplication in RNA seq

3.0 years ago by

United Kingdom

azadjali • 0 wrote:

I am new in bioinformatics and in Galaxy too so please bear with me

Soon I will have RNA seq samples (single reads) for wild type and mutant, and my target is to get expression levels between them.

This is my pipeline :-

Groomer → Filtration (clipping, filtration quality)→ Alignment (Tophat)

↓

Removing of Duplicates (using Picard:MarkDuplicates)

↓

Annotation with the result (Cufflinks) → merging WT& Mutant samples (Cuffmerge) → differential expression (Cuffdiff).

I have taken some samples (H1hsec Rep1, H1hsec Rep2 and Cd20 Rep1, Cd20 Rep1) from Data Libraries to test them as my pipeline

I have few questions about the duplicates - although there is no tutorial showing how to remove duplicates using Picard:MarkDuplicates.

1- Does it required

2- while testing I regularly check every result with fastqc- the percentage of sequences remaining if deduplicated was 19.14% after filtration, while after using picard the percentage of sequences remaining if deduplicated was 3.33% does this mean- it is good

3- Does normalization of the reads automatically happened during application of cuffdiff

Thank you in advance

Regards

Abdul

galaxy • 2.1k views

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modified 3.0 years ago • written 3.0 years ago by azadjali • 0

3.0 years ago by

Jennifer Hillman Jackson ♦ 25k

United States

Jennifer Hillman Jackson ♦ 25k wrote:

Hello,

Expression analysis with this tool suite does not need a step to remove duplicates. In fact, it will almost certainly skew the results. The rest looks fine and follows the recommended method in the manual. Running Fastqc between steps is optional, but if it is helping you, it doesn't change the result.

Best, Jen, Galaxy team

ADD COMMENT • link written 3.0 years ago by Jennifer Hillman Jackson ♦ 25k

3.0 years ago by

azadjali • 0

United Kingdom

azadjali • 0 wrote:

Thank you for your Kind reply dear Jennifer

I think I will follow the same steps in the recommended method [Cufflink version < 2.2.0 still supported]

I wish Galaxy have tools for Cummerbund and shows some videos how to run it

thank you again

ADD COMMENT • link written 3.0 years ago by azadjali • 0

Glad that helped. For more help resources, see the GalaxyProject Vimeo channels and Wiki (Support, Learn, NGS101 and more).

For example, cummerbund (available in the Tool Shed for use in a local or cloud Galaxy) is covered in NGS101-16 at Vimeo, but the associated wiki and videos leading up to it will set the context.

ADD REPLY • link written 3.0 years ago by Jennifer Hillman Jackson ♦ 25k

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