Spec sheet for sequencer-software-kits-shipment-07222021.docx

Specifications of Sequencing and Assembling the Genomes of Three Cultivated and Wild Barley for Disease Improvement

1. General description The barley and oat research team, at USDA-ARS Small Grains and Potato Germplasm Research Unit at Aberdeen, Idaho, plans to sequence and assembly the whole genomes of three barley species to characterize and exploit the unique genes associated with good tolerance to biotic and abiotic stresses and other desirable traits in the landrace of cultivated barley and wild barley species. The genomic data sets will be used to clone disease resistance genes and to develop molecular markers for transferring important genes from wild species to cultivated barley germplasm. The genomes will also be used to conduct comparative analysis and understand the divergence and evolution of barley species.

2. Definitions QC – Quality Control; bp: base pair; Gb – Gigabase; POC – Point of Contact; PE – Paired End; DNA - Deoxyribonucleic acid; ONT: Oxford Nanopore Technologies; HMW DNA: High molecular weight DNA.

3. Tasks and technical requirements for contractor Tasks

1) Conduct genomic DNA quality analysis.

2) Prepare whole genome libraries for Illumina and Nanopore sequencing.

3) Generate raw short-read whole genome sequencing date (≥ 300 Gb for each of the three samples).

4) Generate raw long-read whole genome sequencing date with ONT sequencer (≥ 300 Gb for each of the three samples).

5) Conduct Plant and Animal Genome Survey-Standard Analysis.

6) Combine the NovaSeq PE150 and ONT long-read data sets and generate the assembled genome for each of three samples.

7) Technical requirements

1) Contractor must have laboratory staff experienced in assaying the quality of sequencing libraries and running the Illumina instruments and ONT sequencer.

2) Contractor must have extensive experience and the bioinformatics skills in sequencing, analyzing big genomic data sets and successfully conduct de novo genome assembly or different organisms.

4. Specific requirements for contractor

4.1. Illumina sequencing

a. Must sequence the whole genomes of three plant samples.

b. Must generate ≥ 300 Gb of short-read raw data for each of the three plants.

c. Conduct whole genome library preparation (350bp).

d. The Q30 of PE150 ≥ 85%.

e. Must finish the works above (a-d) in 42 working days starting after receiving confirmation of the sample QC report from customer.

4.2. ONT sequencing

a. Conduct Nanopore DNA library preparation for all three genomes

b. Must sequence the whole genomes of three plant samples with ONT sequencer.

c. Must generate ≥ 300 Gb of long-read raw data for each of the three plants.

d. Must use “High Accuracy (HAC) basecaller model” for read calling.

4.3. Genome assembly

a. Must conduct Plant and Animal Genome Survey-Standard Analysis including:

*Data quality control: filtering reads containing adapter or with low quality *K-mer analysis *GC content analysis *Repeat content rate evaluation *Heterozygous rate evaluation

* Genome size evaluation

* Contamination Inspection

b. Must conduct de novo genome assembly for all three plant samples.

c. Must correct the long-read sequencing errors with the short-read data sets for all three plant samples.

5. Deliverable / Schedule

1) DNA will be submitted to the contractors on an as-needed basis via delivery service throughout the performance period.

2) The submitted DNA sample will be QC analyzed and converted into sequencing libraries and sequenced to the customers specifications as described in the contractor’s submission documentation.

3) All reports will be delivered via email to the government POC, Dongying Gao at Dongying.gao@usda.gov.

4) All final sequencing data, which include a) short-read raw data sets for all three plant samples; b) long-read ONT raw data sets for all three plant samples and c) assembled genomes for all three plant samples, will be provided via a download link.

5) The contractor must send the genome assembly reports including the information about the methods, software and others that were used for the genome sequencing and assembling for all three plant samples.

6) The contractor must send the final sequencing data within 125 WORKING DAYS starting after the contractor receives confirmation of the sample QC report from customer.

6. Security Requirements Data submission portal will be password protected.

7. Data Rights The data will be solely owned by the government and the contractor will have no rights or obligations to the data once it is transferred.

8. Performance Requirement Measures

1) Ensure data quality has a Q30 rating of more than 85%.

2) The contractor should assemble about 85% or more of genome sequences onto seven pseudomolecules for each of the three samples. However, due to the highly repetitive property of the genome (may contain over 70% transposons), lower percentage of chromosome-scale sequences may also be accepted but MUST over 80% of the genome sequences will be assembled into chromosomes.