NGS_target_capture_project_specifications_9-10-18.docx

Specifications for Next Generation Target Sequence Capture in Wheat USDA-ARS is targeting a seven Megabase pair (Mbp) region on chromosome 2BL in 24 wheat (Triticum aestivum L.) samples in order to identify molecular variants closely linked with a stem rust resistance gene. This project will entail purchasing target capture probes for the region of interest, capture library preparation, library pooling and qualification, next generation Illumina sequencing (NGS) and bioinformatics analysis.

USDA-ARS will supply DNA that meet Awardee quantity and quality specifications.

We need a service molecular laboratory that can perform the following:

1. Design and purchase capture probes and associated reagents for a 7 Mbp region on wheat chromosome 2BL in consultation with the USDA-ARS. Capture probes should be comparable to those made by Roche KapaBiosystems and include the following:

a. SeqCap EZ Developer Reagent

b. SeqCap EZ Prime Developer Probes for four reactions

c. HyperCap Target Enrichment Kit for 24 reactions

d. HyperCap Bead Kit for 24 reactions

e. KAPA Hyper Prep for 24 reactions

f. KAPA DI Adapter Kit 15 uM

2. Assess and quantify 24 genomic DNA samples provided by USDA-ARS with a high sensitivity AATI fragment analyzer.

3. Prepare a target capture library for the 7 Mbp genomic region across the 24 genomic DNA samples using the capture probes developed in step 1.

4. Capture and wash the library.

5. Amplify and quantify the capture library using magnetic beads and a high sensitivity AATI fragment analyzer.

6. Capture and pool the libraries for sequencing on an Illumina HiSeq 4000 using bead-based size selection, and library qualification with an AATI high sensitivity fragment analyzer, and qPCR.

7. QC, quantitate and sequence the pooled libraries. QC will be performed with an AATI fragment analyzer, quantification will be performed via qPCR and sequencing will be performed on a HiSeq 4000 to provide 100X coverage of the 7 Mbp region on 2BL and at least 100 Gigabytes of FASTQ sequenced data.

8. Perform custom bioinformatics for wheat (Triticum aestivum L.) target capture using 100 GB of FASTQ sequenced data. Specifically, find SNPs, insertions, deletions and other molecular variants associated with the resistance phenotype in wheat. Wheat is a polyploid with three subgenomes, and a standard bioinformatics analysis may not be sufficient to find variants that amplify from only the subgenome of interest, 2BL.

9. Deliver the final analysis to include raw data files and pertinent sequence data and molecular markers to the USDA-ARS Cereal Pathology Lab in Aberdeen, ID.