Question

Metagenome Assembly Using Combined Reads Through Flash And Using Abyss For Assembly

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10.9 years ago

Sanjiv Kumar ▴ 20

I am using Abyss for assembling paired end Illimuna metagenomic data after comparing forward and reverse reads in two different files and left owers in two other seperate files as follows:

abyss-pe -j10 k=41 n=2 name=Assembly_1 lib='lib1' lib1='../Common_forword_1.fq ../Common_reverse_2.fq' se='../Forword_unique_1.fq ../Reverse_unique_2.fq'

I got the assmebled contigs.

Now when I wish to join the common reads in forward and reverse reads and give it to Abyss for assembly to check if there's some imporvement over the one I already gave.

I have combined all common reads in forward and reverse reads in a single file, concatenated with flash not combined reads and unique forward and unique reverse reads and gave abyss to assemble.

abyss-pe -j10 k=41 n=2 name=Assembly_1 se='../K5_Flash_Combined_NC_FU_RU.fastq'

But the problem is that when I give it to Abyss, it starts assembly, compleats it but dosen't give any contige file.

I was wondering why? Any suggestions will be helpful. Am I doing it wrong? I am new to this so please suggest anything I can try. Thanks a lot.

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ADD COMMENT • link updated 9.4 years ago by Biostar 20 • written 10.9 years ago by Sanjiv Kumar ▴ 20

score 2 · Answer 1 · 2014-02-12

I'm very confused by your methodology here -- not against using ABySS, which is a great assembler -- but just wondering if you're going about the analysis in the right way (I say this as someone who's research focus is metagenomics and metagenomic assembly is a major research question right now). I don't know the ABySS flags off the top of my head, so I'm not sure why you aren't getting any output to your contigs.

Metagenomic assembly chimeras are a major problem and it does not seem that your strategy begins to address the possibility of assembly chimeras in comparing assemblies. You also haven't provided us with any information on the complexity of your metagenome -- human gut microbiome versus soil is a very different problem -- due to taxonomic complexity, presence of microbial strains, different layers of diversity, etc. etc.

There are lots of factors going into assembling metagenomic reads, too many aspects to go into here. I would suggest, for starters, that you work through this tutorial either with your data or the data provided in the tutorial.

score 0 · Answer 2 · 2014-02-17

Tony Raymond suggested:

If you specify the paired end reads with the in parameter, everything should work. The se parameter is meant for single end reads like the ones that are merged with FLASH.

so I issued following command

nohup abyss-pe -j10 k=61 n=2 name=Assembly_1 se='../../Flash_k5_Not_Combined_1.fastq ../../Flash_k5_Not_Combined_2.fastq ../../Forword_unique_1.fq ../../Reverse_unique_2.fq' in='../../Flash_k5_Combined.fastq' &

but that gave following error abyss-fixmate: error: All reads are mateless. This can happen when first and second read IDs do not match. error: `Yam_1-3.hist': No such file or directory

Someone by the name Phillip suggested me to use the following

-se denotes "single end" libraries. You don't want to combine all 3 files. You want to use the common reads for "se" and the two non-merged read files for "lib".

and I issued the flowing command to Abyss for assembly

nohup abyss-pe -j10 k=61 n=2 name=Assembly_1 lib='lib1' lib1='Flash_k5_Not_Combined_1.fastq Flash_k5_Not_Combined_2.fastq Forword_unique_1.fq Reverse_unique_2.fq' se='Flash_k5_Combined.fastq' &

And this worked.

This is for the records, it might help some other newbie like me. Thanks a lot everyone :)