Hi all,
I am a newbie to metagenomics and it is often very confusing on how to analyse my data. I have used the Illumina NextSeq (2 x 150) to sequence a microbial community.
I have used fastQC and trimmomatic for the quality control, and I have assembled the sequences using IDBA-UD. In IDBA-UD, I used command mink=20, maxk=100 for constructing de Bruijn Graph.
There are a lot of output files namely (contig-20.fa, contig-40.fa.....contig-100.fa, contig.fa and scaffold.fa). I would like to do functional annotations and maybe later binning.
Here are the questions:
Thanks for your time on reading my question, if you need anything to be clarified, please let me know.
Cheers and many thanks
Alan
Thank you Asaf.
I was in the IDBA google group but someone suggested using contig.fa as "Scaffold file gonna have lots of Ns (not useful for alignment)" https://groups.google.com/forum/#!topic/hku-idba/D8D46jDjXHE . She suggested that using contig.fa is better for performing BLAST.
Is scaffold.fa better than contig.fa for binning?
Should I use contig.fa for annotation?
Cheers
Alan
It's pretty low... An average protein is 1000 bp long so half of the assembly will contain fragmented proteins. You can check the number of N's in the sequence itself, you can also compare N50 of the contigs to the scaffolds. You can try and assemble with metaSPAdes 3.9.0, it should give better results.
Yeah that was just from the log file of IDBA-UD.
I just ran QUAST on the scaffold.fa and it states n50=1942 while the n50 of contig.fa is 1540. This is the contigs generated from mink=20 and maxk=100.
I ran IDBA-UD again setting mink=100, maxk=121, and Quast shows that n50 of scaffold.fa rises to 3334. However at the same time number of contigs decreased 10 times. (from 52k to 6.1k)
N's per 100kbp ranged from 32 to 38 for scaffold.fa
I will try running IDBA again with mink=60 and maxk=124 to see what I can get.
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