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Old 11-24-2016, 08:10 PM   #1
hamcan
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Default Adaptor Removal with Trimmomatic

Hi there,

I ran a HiSeq2500 run and found that I had adaptor sequences still present.
I first used Trimmomatic to simulatenously remove the adaptor sequences and trim the sequences to a good quality score.

I want to merge the reads next but because I used the Pair-ended format for trimmomatic, I end up with 4 output files: forward paired, forward unpaired, reverse paired, reverse unpaired. How do I go about merging these files? Should I discard the unpaired files and focus on the paired? Or merge the paired together and then concatenate the unpaired? Thank you!
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Old 11-24-2016, 10:57 PM   #2
Zapages
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All you need is Forward_Paired and Reverse_Paired reads. Those reads can be merged together using FLASH, a custom script (there are few here or on Biostar), or you may use Cyverse's Discovery Environment to Interleave the files together into one file.

The unpaired reads can be left alone for now, but in the future you may go back to them to reduce the number of them and so forth by playing around with the trimmomatic settings.

I hope this helps and all the best with your project.

Also check here for more tools that could help you out: https://omictools.com/ with your project.
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Old 11-24-2016, 11:01 PM   #3
hamcan
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Quote:
Originally Posted by Zapages View Post
All you need is Forward_Paired and Reverse_Paired reads. Those reads can be merged together using FLASH, a custom script (there are few here or on Biostar), or you may use Cyverse's Discovery Environment to Interleave the files together into one file.

The unpaired reads can be left alone for now, but in the future you may go back to them to reduce the number of them and so forth by playing around with the trimmomatic settings.

I hope this helps and all the best with your project.

Also check here for more tools that could help you out: https://omictools.com/ with your project.
Thank you so much for your response! After I merge should I concatenate the unpaired files to the merged files? I'm running a BLAST to referenced bacterial genomes.
Another question, for removal of adapter sequences did I need the reverse complement sequences of the nextrra adapter sequences that were provided?
Thank you!
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Old 11-25-2016, 07:10 AM   #4
Zapages
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Quote:
Originally Posted by hamcan View Post
Thank you so much for your response! After I merge should I concatenate the unpaired files to the merged files? I'm running a BLAST to referenced bacterial genomes.
Another question, for removal of adapter sequences did I need the reverse complement sequences of the nextrra adapter sequences that were provided?
Thank you!
I would leave the unpaired files alone.

It all depends on what type of analysis are you trying to conduct?

If you are working on De-novo or novel assembled genomes.

Then the foremost step would be to do a de-novo assembly or do a reference guided assembly of your raw reads into contigs.

After contigs are formed then try to annotate them through BLASTX against a specific database of your choice ie. NCBI Bacterial database, NR Database, or a reference bacterial genome in your case.

If you are doing transcriptome analysis or a de-novo transcriptome analysis

De-novo Assemble your Raw Reads. Annotate your de-novo Assembled reads via BLAST just like mentioned above, and then map them against your reference genome or database of choice (NCBI NR database or Bacterial Database).

If its not a de-novo transcriptome analysis then just map them against your reference genome with the proper GTF/GFF3 files and settings.

Hope these strategies help.

Last edited by Zapages; 11-25-2016 at 07:12 AM.
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