Platforms/strategy for sequencing of new genome/resequencing of a known genome

[rameshka]

Hi everyone!!

I am happy to join this great forum, especially as I plan to go for NGS based sequencing in our research; here, I may admit that I am a complete novice with using NGS, but we plan to use these powerful approaches to sequence: a new unknown genome (of ~ 600 MB size), and reseq 2 cultivars of another plant species for which a draft genome sequence of ~750 MB is available.

I will be grateful for any help/suggestions on: desired NGS platforms/strategy for the above two studies.

Cheers..

Rka

[krobison]

Your first requirement is to clearly enumerate and rank the goals of the study and also what your budget is.

While your project is another notch up in genome complexity, a good starting point is this recent thread.

[rameshka]

thanks Krobison for the inputs, and providing the link to Lex Nederbragt's recent blog post on sequencing that is very informative..

our goals are as follow:

1. de novo sequencing of a new plant genome of ~600Mb size for which presently, no reference is available.

2. re-sequencing two parental genotypes of another plant species for which whole genome sequence (~750 Mb) is available; idea is to generate genome reconstruction of our genotypes, and also to develop SNP arrays based on polymorphism between the two parents for use in mapping studies.

For the 1st one, we presently has a vague plan comprising- a low (~10x coverage) using Ion-Torent or 454 runs, and then have a high coverage using Illumina paired-end seq data ..

for the 2nd study, we intend to go for 20-30X coverage by re-seq using Illumina platform.

we have the 454 and IonTorent platforms available with us, and plan to generate our own data/as well as outsource some of it; but bioinformatics analysis, we definitely would need to outsource ..

budget available is not high but in the range of 30-50K US$

best..

[krobison]

Sorry, I wasn't very clear. By goals I mean the scientific questions you wish to answer. For example, getting a rough gene census, enumerating SNPs or other variants, etc. These will drive the requirements for your sequence quality.

It's not obvious to me that Ion Torrent would add anything to your first project & it's expensive to get deep enough 454 coverage. Really deep Illumina paired end coverage will get you a good first look, and it will be rare that low coverage on the other platforms will get you much farther.

For <$30K outsourced, you could run all three on an Illumina HiSeq flowcell (with library construction) as 2x100, and still have lots of room for some mate pair libraries in the same run. Your remaining $20K (high end of your range) might get you decent coverage with long PacBio reads, or you could use that for optical mapping.