2019-8-14 Meeting Notes EiB Polyploid Working Group

Started with SolCap Illumina array (~8,000 markers) and selected polypmorphic markers approx every 1 cM. Four trait based KASP markers were also included. 

Started with sending 1,500 markers to DArT

Hannale provided CIP populations and high quaility reference genome DNA and Jeff provided some NA germplasm - DArT went through 2 cycles of selection ending with 855 markers. Only 1 of the trait based KASP markers was in the final set, but the others can be tweaked if needed. Sequence depth was very even across markers (screamers were removed) at ~ 800 reads per locus. This is considered to be 'obver sequenced' and in production this will likely be reduced to meet estimate costs/sample.

Jeff thinks that read depth could be as low as 10 per sample with the new imputation methods they have for dosage

Repeatability between sample reps was very high - r(2) 0.98-0.99

Each amplicon has between 60-80 bases - is this enough to call haplotypes?Jeff thinks so. 

Andrzej says he can deliver data formats in what ever is the community standard but he can't accomodate multiple formats  - sending fatqs will be extra ($200?)

 Andrzej – review data

Controls are G12 and H12 for DArT and Intertek can handle this

In cassavabase have an ordering form to create Intertek and DArT orders

DArT can provide a vcf as well as tab delimited

Have a script to convert vcf to genotyping files

In cassavabase will use vcf dosage data in GS ie user doesn't have to do anything

Have loaded 20,000 samples for DArT-LD already

Polyploids - can store dosage in cassavabase but not sure how to handle downstream tools

Elisa - can Guillaume share ontologies with potato base for molecular markers? Maybe have a follow up meeting on this? eg protocols, platform 

DArT-LD get 6-10,000 markers with good coverage - approx 20% missing data. Loading data as-is and imputing.

Dorcus - used DArT-seq for sweet potato. Ask for raw fastq files and use this for polyploid calling. Using a different vendor too optimized for polyploids. 

Jeff - need allele depth - need to communicate to DArT. Top on wish-list for Guillaume too. 

Dorcus - when call data with dosage the quality is not very high - need 3-4 times higher depth

For Yam just used the standard protocol

Jeff - have used intertek for KASP for potato - results were consistent with in-house results. For genome wide markers = have provided a set of SNPs for primer design 1,200-1,300 primers ordered. Waiting for wet-lab to see which will be working

Jeff - Dart wants the DNA of reference genome to set up for internal QC for development of plex. 

Guillaume - 100 bp sequence each side and they design sequence.

Correct and complete order form - will be generated by Sample Manager in future

Shipments of multiple of 16 x 96 plates. 

Agreed quality and quantity of sample

Agreed plasticware - Rajaguru would have this info and we can send it out 

Fixed plate layouts 
Payment via Charge account

Agreed SNPs with VERY GOOD Assessment class (for KASP)