Hi all,
I need to analyse results coming from next generation sequencing on bacterial 3 strains that are carbapenem-resistant. I am using the software "Artemis" to analyze the sequences. I anticipate that this stuff is totally new for me. I am particularly interested in the resistance genes and in the determination of their genetic environment. I was wondering how I can understand from the sequencing data if the resistance genes are on plasmids or on the chromosome?How can I distinguish plasmids and chromosome by the sequencing data?What kind of problems or issues can I encounter when I analyze the data?Is there anything to which I should be more careful in the analysis of the data (e.g. could the sequencing method create contigues that don't correspond to the real aspect of the genome)?
Thanks
After assembly with certain short read assemblers (Abyss, for example), the contigs corresponding to plasmids exhibit the same sequence at their termini. Another thing to look for is the presence of plasmid-specific replication origins or replication-associated protein coding sequences.
Ok. Please, many concepts are completely new. I have never done something like this.
Can you clarify me the concept of coverage in the sequencing?I found the definition of :
"Coverage (read depth or depth) is the average number of reads representing a given nucleotide in the reconstructed sequence" . I can't figure out. Is this the number of times a same nucleotide appears in the reads??Can you give me some practical examples and explain me in simple words?
I attached a figure showing what I visualize when I open the results of sequencing of my bacterial strain with Artemis. Please, can you help me with the interpretation of data? what does the first line "source" refer? Is it a single contig? why is it not reported as "CDS", similarly to the others?and the number reported is the size of the contig?All these CDS I obtain, therefore, if I understtod correctly, are different pieces of the sequence ...aren't they?
My bacteria should have resistance genes in its plasmids(beta-lactamases), as obtained by PCR. Anyway I tried to open the "CDS genes and product" in Artemis that gives (in a text file) all the genes codifying the proteins found. Nevertheless, I can't find any CDS for the beta-lactamases I am looking for ...why does it happen? How can I retrieve the sequence of them? I am sure they are in the sequence of the bacterium..Please, HELP!
Dear Silvia,
i have no experience with Artemis but if you want to find your resistance genes you can submit your contigs ( sequencing data) to Resfinder ( online free tool for finding resistance genes) https://cge.cbs.dtu.dk/services/ResFinder/
There you will get out in which contig the genes are found and where in the contig. AS for coverage: if you have your consensud sequence from al your reads or contigs- you can map all reads to this consensus sequence and find how many times each nucleotide is covered by the reads. If you have two reads spanning one region this region(or nucleotides in this region) have a cobverage of 2 and so on.
Good luck!
Best Jane
I am not sure what your "sequencing results" file looks like. But I have gotten sequencing results files from a sequencing and annotation service that contained hundreds of contigs all in one file. When I open that file with Artemis I see only the first entry and no indication that the file contained hundreds of contigs.
There are several ways to split the multi-entry file into one entry per file:
http://seqanswers.com/forums/showthread.php?t=18525
but I am not sure you want that. You may want only the contig(s) that contain the beta lactamase gene(s) singled out and not jam up your file space with hundreds of individual files. For my work, it was easy to locate the few contigs I was interested in and split them out into separate files while leaving the main multi-entry file intact.
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