1116802 Altendorf Sequencing SOW.docx

Statement of Work (SOW) Project: Hops NAM Population Sequencing Project

The USDA-ARS hops breeding program in Prosser, WA has identified 576 hops plants to be sequenced to aid in the discovery of novel genetic resistance to powdery mildew.

Vendor will receive 30 mg freeze dried leaf tissue of 672 samples via mail from Customer in 96-well plate format (Qiagen Catalog #19560 or Corning Life Sciences #4412) in 7 plates. Vendor will also receive an Excel file with plate ID designators outlining the sample IDs associated with each well. Each plate will contain at least two blank wells for quality control as outlined in the maps.

The Vendor shall extract high quality genomic DNA, normalize samples and assess quality, create genotyping by sequencing libraries using NspI and BfuCI enzymes, and genotype all Customer samples using a Life Technologies’ Ion Proton System, or similar. Samples with insufficient DNA quality or quantity shall be reported to the Customer and the opportunity shall be provided to send additional tissue or DNA. Samples will be genotyped using a tune-able Genotype-by-Sequencing (tGBS) method. tGBS is a type of sequencing for plants that produces very high-quality genotyping for relatively low cost with parameters that can be tuned for optimal genotyping in heterozygous non-model plant species such as hop. Sequencing should produce an average of 2,779,066 reads per sample (range = 1,069,794 – 6,838,292), with an average read depth of 38. The Vendor shall use the Cascade Genome (https://hopbase.org/dovetailDownloads/dovetailCascadeMasked.php.) sequence to align reads with quality score greater than 15, unique alignments, and call SNPs using the following parameters. For homozygous SNPs: The most common allele must be supported by at least 80% of all the aligned reads covering that position, at least 5 unique reads must support the most common allele, polymorphisms in the first and last 3 base pairs of each read are to be ignored, and each polymorphic base must have at least a PHRED base quality value of 20. For heterozygous SNPs: Each of the two most common alleles must be supported by at least 30% of all aligned reads covering that position, at least 5 unique reads must support each of the two most common alleles, the sum of reads of the two most common alleles must account for at least 80% of all aligned reads covering that nucleotide position, polymorphisms in the first and last 3 base pairs of each quality trimmed read are to be ignored, and each polymorphic base must have at least a PHRED base quality value of 20. A range of 2,000 – 10,000 markers shall be reported in vcf file format.

The Vendor will provide the following: 1) raw sequence data in the format of fasta files; 2) an unfiltered vcf file of marker data from and 3) any remaining frozen DNA samples from library development in plate format; within 8-12 weeks from receipt of Customer’s leaf tissue samples. Data will be made available to Customer via download link and via hard drive which shall be sent in the mail.