SOW_date_palm sequencing.docx

US Department of Agriculture (USDA)

Statement of Work

Contents

Project Date Palm Sequencing and Annotation

General Information

1.0 Scope of Work

The objective of this project is to assemble de novo sequences of two date palm cultivars and annotate the assembly using RNA-seq data.

2.0 Background

We are beginning a project to evaluate differential gene expression (DGE) for fruit of date palm (Phoenix dactylifera L.) of two different cultivars, one of which (‘Medjool’) experiences a physiological phenomenon called “puff”, the other of which (‘Deglet Noor’) does not. This condition is expressed in reaction to specific environmental conditions at a specific fruit development stage. We hope to gain an insight to the genetic basis for these differing responses as well as other processes during fruit development.

Contractor Requirements

3.0 Technical Requirements / Tasks

DNA: The contractor shall extract high molecular weight (HMW) DNA (fragment size of 50 – 100 kb) from a young leaf samples of P. dactylfera submitted by the Government.

Library Preparation and Sequencing: The contractor shall generate the number of SMRT cells required to generate ~30X HiFi sequence coverage using HiFi PacBio technology. This is based on the genome size provided by the Government (~670 Mb to 772 Mb and 18 chromosomes).

Library Preparation: The contractor shall construct Omni-C libraries to target 15X coverage per haplotype of PE150 reads. The contractor shall sequence ~2M PE 75bp reads and map the data back to the draft assembly to assess library quality.

RNA Sequencing Library Preparation: The contractor shall extract RNA and prepare a standard RNA library with rRNA depletion. 2 x 150bp reads shall be sequenced on an Illumina platform to the target of ~20M read pairs per sample.

Assembly Informatics Package: The contractor shall scaffold the draft assembly (minimum N50: 100 kb) through a Dovetail HiRise software pipeline using Omni-C library data.

The contractor shall perform TAD analysis on the genome generated from Omni-C libraries if 40% of read pairs should map at a distance of greater than 1 Kbp, genome size is less than 10 Gb, assembly scaffold size is less than 400 Mbp, more than 40% of the genome is uniquely mappable, and coverage is 100 M read pairs per 1 Gb.

Gene Annotation: The contractor shall annotate the whole genome annotation of a genome assembly using RNA-seq data using tissue submitted by the Government (6 different tissue types). The assembly must have more than a 70% BUSCO single copy or duplicated score. The contractor shall provide annotation service including repeat masking, ab initio gene prediction using related species, RNA seq mapping to enhance annotation, manual curation of 5 genes, and assignment of functional tags to genes.

4.0 Government Furnished

The government will furnish leaf tissue of the two date cultivars in quantities sufficient for the contractor to perform the work. The Government shall further furnish tissue from date palm leaves, roots, flowers, embryos, fruits, and seeds in sufficient quantities for whole genome annotation.

5.0 Deliverables / Schedule

The contractor shall furnish within 9 months the following:

• HiRise Assembly deliverables:

• The HiRise assembly in FASTA format

• A report summarizing key assembly statistics, features of the Omni-C library, and a linkage density plot of the proximity ligation library data

• A table detailing the breaks made to the input scaffolds

• A table describing the position of the input assembly scaffolds within the final HiRise scaffolds

• BAM file(s) containing alignments of the Omni-C library read pairs mapped to the draft assembly

• TAD Analysis deliverables:

• TAD calls at 10kbp, 25kbp, and 50kbp

• CTCF binding sites

• A/B compartmental organization

· Isochore locations

· Guide to loading and viewing data in HiGlass

• Summary report

• RNA Sequencing Library deliverables includes six (6) FASTQ files, each containing ~20M read pairs.

• QC report

• Raw ILMN data from deep sequencing the Omni-C library

• Genome Annotation deliverables shall consist of a comprehensive report including:

• Genome annotation file in GFF3 format

• Predicted gene CDS sequences in FASTA format

• Predicted gene peptide sequences in FASTA format

• Alignment files from RNA-seq and Iso-Seq data in BAM format (if evidence data is provided)

• Repeat annotation file in GFF3 format

• Visualization of structures of manually curated genes

• The polished hifiasm assembly, including:

• The primary contigs in FASTA format

• A report summarizing key data and assembly

· The HiFi PacBio sequence in BAM format

6.0 Travel

N/A

7.0 Contractor’s Key Personnel

N/A

8.0 Security Requirements

N/A

9.0 Data Rights

If data is to be produced, furnished, acquired, or used in meeting contract requirements, delineate the respective rights and obligations of the government and the contractor regarding the use, production, and disclosure of that data.

Insert Data Rights Here

10.0 Section 508 – Electronic and Information Technology Standards N/A

Attachment No attachments.

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