SBT from amplicons

[Mattstorey]

Hi, Great tool for implementing L. pneumophila SBT! Thanks.
I'm trying to call SBT using nanopore sequenced multiplex PCR amplicons. However, I get "no product" results for some of the alleles. Sequences for the alleles are in the input file and are a perfect match for an allele in the database. I tested an assembled genome of the same SBT strain and get expected results. Can I use amplicons with Legsta? Do the input sequences need to be longer?

Cheers!

[tseemann]

If the primers are still in the amplicons it should (probably) still worki? Or do you trim them off?

Are you able to upload the amplcons to the PHE SBT website? Or is it still down.

[Mattstorey]

Hi, Torsten.

The amplicons are primer to primer and map with ~100% concordance to the expected reference. Unfortunately, the SBT website is still down.

Cheers

On Tue, Aug 4, 2020 at 10:16 AM Torsten Seemann notifications@github.com wrote:

If the primers are still in the amplicons it should (probably) still worki? Or do you trim them off?

Are you able to upload the amplcons to the PHE SBT website? Or is it still down.

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[andersgs]

@Mattstorey can you provide a test case for us to try to reproduce the issue?

Thank you. Anders.

[Mattstorey]

Hi, @andersgs

Attached are amplicon seqs. When I run legsta with this file the results are:

FILE    SBT     flaA    pilE    asd     mip     mompS   proA    neuA
Running: any2fasta -q -u SBT_contigs\.fasta | isPcr stdin \/home\/mstorey\/\.conda\/envs\/Legionella\/db\/ispcr\.tab stdout -out=fa -minPerfect=5 -tileSize=6 -maxSize=1200 -stepSize=5
SBT_contigs.fasta       -       3       4       1       1       -       9       -

So some work and some don't in this case.

I did the individual pairwise alignment manually for each gene and got 100% concordance to:

flaA    pilE    asd     mip     mompS   proA    neuA
 3       4       1       1       14       9       1

Which is SBT 36 as expected.

SBT_contigs.fasta.txt

[andersgs]

Thank you @Mattstorey.

We will see what is happening.