Hi all,
I am trying to interpret the results coming from ngs on some bacterial isolates that are antibiotic resistant. This is completely new for me and I have different questions about it.I would like to know; how I can reconstruct the sequence of the plasmids starting from the contigs I have? I mean, I have different contigs..should each contig correspond to a different plasmid? And if not, how can I "re-build" the entire sequence of the plasmid from these contigs?
Then..In order to see if particular genes were present in my isolates, I run Blast by using the sequence of a known gene as a query against the FASTA file containing all the contigs..As a result, I have different contigs that match with the known sequence. Does it mean that I have the plasmid sequence split in different contigs?or anything else?
furthermore, i find the same sequence alignment with different contigs..so it seems like the same sequence is repeated in different contigs (?) what does it mean? That more contigs containing the same sequence can be generated by the sequencing procedure?
Thank you very much
Dear Doctor,
you can follow these steps:
- Contig Assembly DNA Sequencing, using DNA Baser Sequence Assembly Software, link, http://www.dnabaser.com/
- Comparing nucleotide or protein sequences to published GenBank sequence databases using National Center for Biotechnology Information-Basic Local Alignment Search Tool (NCBI-BLAST), link: https://blast.ncbi.nlm.nih.gov/Blast.cgi
- Alignment of the nucleotide and amino acid sequences using MEGA5 program, product version 5.1, link, http://www.megasoftware.net
Ok. I will try.Can I ask why I find contigs that match the same known sequence (when I run Blast) but they show slightly differences in terms of their % identity and coverage with it? For example I have two contigs that match the known sequence for the same region, one matches it with 100% identity , the other one with 99%...Does it mean that normally I have more possible contigs for the same sequence?? on what does it depend? The sequencing was carried out with Illumina
Thanks
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