Fragment analysis; microsatellite or SSR/STR

fadss · 01-10-2014, 02:24 PM

Has anyone been using next gen or preferably illumina for fragment analysis.
Right now my project are running multiplex for Sanger 3130xl machine to perform fragment size analysis.
But this limited to small sample and run 1 sample per capillary and require culture of bacteria for pure cullture.

I wish to know if illumina are limited to only sequencing?
or cant I just sequence the microsatellite loci and look at the length for fragment size of millions different strains and sort them using computer program some how.

So instead of having 10 to 100s sample size; I would have over 100,000 or millions.

but with multiplex(with many loci), how can i separate which strain to which since it all mix.

Thanks,

JackieBadger · 01-10-2014, 02:30 PM

I can't answer your question directly, but yes you can sequence microsats using Illumina. In theory it will provide more accurate allele characterization than bands on a gel.
You can use our recent bioinformatic approach to genotype micros using NGS

http://onlinelibrary.wiley.com/doi/1...12225/abstract

fadss · 01-10-2014, 02:50 PM

basically I have a vial of mix strains of bacteria but a single specie. I wanted to know %proportion of each strain in a time period.

differentiate between strains by pcr-multiplex amplify 5 microsatellite loci of length from 100bp to ~400bp
each strain have different pattern of these fragment size.

right now I am culturing at each time point and and pick out 10 colonies each time.

But what i heard is that next gen dont really need culturing them into media agar plate.

Just looking for alternatives...with luck that it could help with finer resolution in %proportion

bunce · 01-20-2014, 03:58 AM

Hi Fadss, Yes NGS (primarily 454) has been used to type STR's. The main application to date has been the CODIS human ID markers used in forensics. We are yet to try it on our MiSeq but, it should work better than 454 and PGM due to the low homopolymer error.
check out:
Bornman, D., Hester, M., Schuetter, J., Kasoji, M., Minard-Smith, A., Barden, C., Nelson, S., Godbold, G., Baker, C. , Yang, B. 2011. Short-read, high-throughput sequencing technology for STR genotyping BioTechniques 2012.

Fordyce, S.L., Ávila-Arcos, M.C., Rockenbauer, E., Børsting, C., Frank-Hansen, R., Petersen, F.T., Willerslev, E., Hansen, A.J., Morling, N. , Gilbert, M.T.P. 2011. High-throughput sequencing of core STR loci for forensic genetic investigations using the Roche Genome Sequencer FLX platform Biotechniques 512: 127-133.

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01-10-2014, 02:24 PM	#1
fadss Junior Member Location: Britsh Columbia Join Date: Jan 2014 Posts: 4	Fragment analysis; microsatellite or SSR/STR Has anyone been using next gen or preferably illumina for fragment analysis. Right now my project are running multiplex for Sanger 3130xl machine to perform fragment size analysis. But this limited to small sample and run 1 sample per capillary and require culture of bacteria for pure cullture. I wish to know if illumina are limited to only sequencing? or cant I just sequence the microsatellite loci and look at the length for fragment size of millions different strains and sort them using computer program some how. So instead of having 10 to 100s sample size; I would have over 100,000 or millions. but with multiplex(with many loci), how can i separate which strain to which since it all mix. Thanks,

01-10-2014, 02:30 PM	#2
JackieBadger Senior Member Location: Halifax, Nova Scotia Join Date: Mar 2009 Posts: 381	I can't answer your question directly, but yes you can sequence microsats using Illumina. In theory it will provide more accurate allele characterization than bands on a gel. You can use our recent bioinformatic approach to genotype micros using NGS http://onlinelibrary.wiley.com/doi/1...12225/abstract Last edited by JackieBadger; 01-10-2014 at 07:01 PM.

01-10-2014, 02:50 PM	#3
fadss Junior Member Location: Britsh Columbia Join Date: Jan 2014 Posts: 4	basically I have a vial of mix strains of bacteria but a single specie. I wanted to know %proportion of each strain in a time period. differentiate between strains by pcr-multiplex amplify 5 microsatellite loci of length from 100bp to ~400bp each strain have different pattern of these fragment size. right now I am culturing at each time point and and pick out 10 colonies each time. But what i heard is that next gen dont really need culturing them into media agar plate. Just looking for alternatives...with luck that it could help with finer resolution in %proportion

01-20-2014, 03:58 AM	#4
bunce Member Location: Perth Join Date: Sep 2012 Posts: 55	Hi Fadss, Yes NGS (primarily 454) has been used to type STR's. The main application to date has been the CODIS human ID markers used in forensics. We are yet to try it on our MiSeq but, it should work better than 454 and PGM due to the low homopolymer error. check out: Bornman, D., Hester, M., Schuetter, J., Kasoji, M., Minard-Smith, A., Barden, C., Nelson, S., Godbold, G., Baker, C. , Yang, B. 2011. Short-read, high-throughput sequencing technology for STR genotyping BioTechniques 2012. Fordyce, S.L., Ávila-Arcos, M.C., Rockenbauer, E., Børsting, C., Frank-Hansen, R., Petersen, F.T., Willerslev, E., Hansen, A.J., Morling, N. , Gilbert, M.T.P. 2011. High-throughput sequencing of core STR loci for forensic genetic investigations using the Roche Genome Sequencer FLX platform Biotechniques 512: 127-133.