Question: Obtain sequence of a gene with vector DNA integrated
0
gravatar for kspata
6 weeks ago by
kspata40
Chicago
kspata40 wrote:

Hi,

I have 1 plasmid sample sequenced on MiSeq PE150. I have derived the sequence of putative plasmid by de novo assembly (SPADes). The plasmid contains 5' AOX1 primer sequence and reverse complement of 3' AOX1 primer along with possible insert sequence.

I have 2 genomic DNA sequenced on MiSeq PE150. I wish to find partial sequence of the AOX1 gene with vector DNA inserted from these two samples.

I have the 5' AOX1 and 3' AOX1 sequences.

How can I achieve this? Can someone suggest any strategy?

Thanks!!

ADD COMMENTlink modified 6 weeks ago by Vitis2.1k • written 6 weeks ago by kspata40
1
gravatar for Vitis
6 weeks ago by
Vitis2.1k
New York
Vitis2.1k wrote:

Create a new reference genome with the additional plasmid sequences, map the genomic reads to the new reference, then hunt for read pairs that span the genome and plasmid, i. e. read pairs with one mate mapped to the genome and the other mate mapped to the plasmid. There would also be reads spanning the genome/plasmid junctions so you may look for those reads, too. They would appear as clipped mapped reads with secondary alignments.

ADD COMMENTlink written 6 weeks ago by Vitis2.1k

Hi Vitis,

Thanks for quick response!!! I do not have the reference genome sequence. I only have the plasmid sequence. Is there a way to do it without the reference sequence?

ADD REPLYlink written 6 weeks ago by kspata40

Maybe do a de novo assembly with the reads from genomic DNA? Not sure data from two MiSeq runs would be enough. The other option is to try something like Capture-based sequencing, in which probes designed based on the plasmid sequences would capture and enrich the genomic DNA fragments containing the plasmid. This approach is considerably slower and more expensive. Finally, there is the option of using PCR with one primer within the plasmid region and one random primer (like old school genome walking techniques). This is much more noisy as the random primer would give you a lot of non-specific amplifications.

ADD REPLYlink modified 6 weeks ago • written 6 weeks ago by Vitis2.1k

Hi Vitis,

I did perform de novo assembly on genomic data but its is not enough to generate entire genome of the yeast. The coverage is very less.

Can designing primers using the 5' AOX1 region and 3' AOX1 region and performing Sanger sequencing help. As these regions flank the gene, they should also capture the vector DNA. Will this approach work?

ADD REPLYlink written 6 weeks ago by kspata40
1

This should help you determine the presence of plasmid insertion at the intended site, but may not reveal additional insertions that might happen somewhere else.

ADD REPLYlink written 6 weeks ago by Vitis2.1k

I was a bit confused, maybe I missed something. You said it is a yeast? Why couldn't you use the current yeast reference genome, or it is some specific strain without a reference genome?

ADD REPLYlink written 6 weeks ago by Vitis2.1k

It is a specific strain without reference genome.

ADD REPLYlink written 6 weeks ago by kspata40
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