Question: Metagenome Assembly Using Combined Reads Through Flash And Using Abyss For Assembly
gravatar for Sanjiv Kumar
5.1 years ago by
Sanjiv Kumar20
New Delhi, India
Sanjiv Kumar20 wrote:

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.

assembly • 2.8k views
ADD COMMENTlink modified 3.7 years ago by Biostar ♦♦ 20 • written 5.1 years ago by Sanjiv Kumar20
gravatar for Josh Herr
5.1 years ago by
Josh Herr5.6k
University of Nebraska
Josh Herr5.6k wrote:

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.

ADD COMMENTlink modified 5.1 years ago • written 5.1 years ago by Josh Herr5.6k

Thanks for the response and the link.

I did the basic filtering of the data with respect to the quality control. Sorry about the missing information.

The data is from a contaminated sewage water, so should be quite enriched and complex in term of taxonomic complexity, but that's a presumption. You are right in saying that I need to do a bit of more homework with respcet of the choice of assembly process. Abyss was the first one I tried and it seems working fine and I intended to explore variation with hope of improving assembly. I did assembly again providing joined combined FLASH reads as single end reads along with the left overs in seperate file, but with same results, no contigs file. Was wondering how FLASH combined reads are given to Abyss along with the leftovers. Going to try some other assemblers.

Once again thanks for the link, I am out to explore :).

ADD REPLYlink written 5.1 years ago by Sanjiv Kumar20
gravatar for Sanjiv Kumar
5.1 years ago by
Sanjiv Kumar20
New Delhi, India
Sanjiv Kumar20 wrote:

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 :)

ADD COMMENTlink written 5.1 years ago by Sanjiv Kumar20

So is there improvement?

ADD REPLYlink written 5.1 years ago by Adrian Pelin2.2k
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