Entering edit mode
5.2 years ago
kspata
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80
Hi,
I have PCR reaction products obtained using nematode 18S primers and sequenced on Illumina MiSeq PE 250. My aim is to identify and characterize the Nematode species from the NGS data. I do not have the reference sequence for this.
I performed BLAST on a subset of reads which does not give identity to any of the nematode species.
What existing tool/method and database should I use to perform this analysis?
Thanks!!
What did you blast against? At NCBI or local? If local, did you get the taxonomy database file, if you downloaded pre-made indexes from NCBI.
I performed BLAST at NCBI using default parameters against the Other Nucleotide database (nr/nt).
Have you looked at "Formatting options" and then under "Other reports" clicked on "Taxonomy report"? You should see something like this.
I checked the taxonomic report. It shows matches with Eukaryotic synthetic constructs.
Does this mean that the Sample has no matches with Nematoda? Or should I use another approach?
Check the identity of the hits, if this is a high percentage you just sequenced yourself or the lab technician =). This is something that can happen, it is contamination. Try to check some more reads. There is no other tool or method that gives you different result. You just need to blast all your reads en remove the Homo Sapien ones.
Side note: Do not actually just blast all your reads, you need to clean up the files, trim the primers, cluster, chrimera checking and some other steps)
Thank you for replying. I am trying to upload the image without success. I will try Blasting another subset of reads.
I saw the image, needed to select the url from the text but I could see it. This is your link https://i.imgur.com/K0Fod8X.jpg
kspata Use these directions: How to add images to a Biostars post
Also check if the Homo Sapien hit is a 18S sequence or not
blasting is a good approach. Do you have hits on other species or no hits at all?