Detecting Known, Low Frequency SNPs in evolving bacterial communities

jleehan · 01-28-2019, 12:44 PM

Hi all,

I'm using NGS data to observe how a community of congenic bacteria evolve over time. Basically, I'm starting by inputting several different alleles of one gene into the same medium and seeing how that population changes over time by sequencing the DNA of that gene.

As far as I can tell, all the SNP calling pipelines appear to be more for genome wide discovery than something like what I am doing.

Currently, I am using VarScan and LoFreq to determine the allele frequencies in this community. The agreement between the two pipelines is decent, but as of now VarScan doesn't seem to be able to detect samples that are present in less than 20% of the population (despite what was reported in this paper and all the other programs are reported to be even worse) and I would like to be able to use at least two different programs to verify the presence of these alleles.

Does anyone know of a better low frequency SNP calling pipeline or perhaps a program that allows you to call SNPs based on known alleles (this would actually be the ideal, but I haven't seen anything like this anywhere that I've read)?

Thanks!

P.S. I'm relatively new to NGS work so please bear with me if I've made some egregious oversight here.

Meyana · 01-28-2019, 08:52 PM

I have been really pleased with using the variant caller that is a part of the BBTools package for rare variants in mitochondrial DNA.
https://jgi.doe.gov/data-and-tools/b...ls-user-guide/
It is not so well documented at that site, but look here for how to use:
https://github.com/abiswas-odu/Disco...allvariants.sh
or throughout this forum.

jleehan · 01-29-2019, 08:06 AM

Thank you very much for your recommendation, I'll be sure to check it out!

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01-28-2019, 12:44 PM	#1
jleehan Junior Member Location: Florida Join Date: Jan 2019 Posts: 3	Detecting Known, Low Frequency SNPs in evolving bacterial communities Hi all, I'm using NGS data to observe how a community of congenic bacteria evolve over time. Basically, I'm starting by inputting several different alleles of one gene into the same medium and seeing how that population changes over time by sequencing the DNA of that gene. As far as I can tell, all the SNP calling pipelines appear to be more for genome wide discovery than something like what I am doing. Currently, I am using VarScan and LoFreq to determine the allele frequencies in this community. The agreement between the two pipelines is decent, but as of now VarScan doesn't seem to be able to detect samples that are present in less than 20% of the population (despite what was reported in this paper and all the other programs are reported to be even worse) and I would like to be able to use at least two different programs to verify the presence of these alleles. Does anyone know of a better low frequency SNP calling pipeline or perhaps a program that allows you to call SNPs based on known alleles (this would actually be the ideal, but I haven't seen anything like this anywhere that I've read)? Thanks! P.S. I'm relatively new to NGS work so please bear with me if I've made some egregious oversight here.

01-28-2019, 08:52 PM	#2
Meyana Member Location: Japan Join Date: Sep 2017 Posts: 40	I have been really pleased with using the variant caller that is a part of the BBTools package for rare variants in mitochondrial DNA. https://jgi.doe.gov/data-and-tools/b...ls-user-guide/ It is not so well documented at that site, but look here for how to use: https://github.com/abiswas-odu/Disco...allvariants.sh or throughout this forum.

01-29-2019, 08:06 AM	#3
jleehan Junior Member Location: Florida Join Date: Jan 2019 Posts: 3	Thank you very much for your recommendation, I'll be sure to check it out!