Reference gene identification for contig ordering, when sample species is unknown

Question

[shuksi1984]

How do I choose a reference genome to order the contigs once after being assembled? in my case, I have no information about clinical isolate of the microorganism. Therefore, based on what do I choose a reference for conting ordering using Mauve. My sample is "ERR209055" downloaded from EBI and task is "antimicrobial resistance gene identification".

Answer 1

[gb]

I checked ERR209055 and it is a human gut metagenome. So it is a mix of a lot of different species, and therefore you can not choose a reference. Why do you need to order the contigs? After assembly you need to predict the open reading frames and blast it against a antimicrobial resistance gene database.

[shuksi1984]

you can not choose a reference

In the above case, if I have a human sample with E.coli, how to choose the reference?

you need to predict the open reading frames

How do I do that?

[gb]

Depends on what your goal is I think. But if it is only human and E.coli you could use human and E.coli. You could also only use E.coli if you are not interested in the human genes. I personally think that if the goal is to find antimicrobial resistane genes you dont have to worry to much about human genes. But you said you already had done a assembly.

For predicting genes there are many tools: https://en.wikipedia.org/wiki/List_of_gene_prediction_software

[shuksi1984]

How do I choose raw data from ENA, to find anti-microbial resistance genes and multi-loci sequence typing. Can I select human isolate containing single microorganism or multiple microorganism species? I choose "SRR1060710" for both the above task. Am I doing right?

[gb]

Just to test or to try to find those genes it does not matter if you choose data from a single microorganism or multiple microorganism (metagenome). It helps if you know upfront that the organism has shown antimicrobial resistance in other studies. Globally:

1. download the raw fastq files
2. Do a assembly, denovo or reference based
3. Find the open reading frames (gene prediction)
4. Blast it against known antimicrobial resistane genes

Most resistance proteins consist of multiple domains. You often find a gene that codes for one domain but for resistance multiple domains are needed.