Attachment_1-_Performance_Work_Statement.docx

PERFORMANCE-BASED WORK STATEMENT

I. GENERAL INFORMATION

A. Introduction

The U.S. Geological Survey is seeking DNA library preparation, next generation sequencing and custom reference library preparation for analysis of environmental DNA samples. The project will require assay optimization, sample preparation, sequencing, and bioinformatics analysis of DNA sequences of various vertebrate and invertebrate species.

B. Background

The PI is investigating the presence of invasive and native vertebrate and invertebrate species across the United States by developing environmental DNA (eDNA) metabarcoding assays. The PI anticipates metabarcoding sequencing of roughly ~200-300 samples for the year of the award. As eDNA samples are often fragmented and as the DNA is being isolated from water or swabs as opposed to tissue or blood, the contractor shall perform analyses using super high accuracy base calling and a specialized sequencing pipeline to maximize efficiency.

The DNA will be used to construct libraries, which will then be PCR amplified and sequenced using 2-step PCR with high sensitivity (6 PCRs pooled), and super high accuracy base calling. Raw data is then transformed into sequenced reads and quality filtered before being entered into the specialized bioinformatics analysis pipeline. A custom reference library will be developed and used to identify high-quality exact sequence variants (ESVs) sequence data. Taxonomic assignments, ESVs, Fastq and QC files will be delivered to the PI in a secure manner.

C. Definitions/Applicable Documents: Not required

D. Scope The period of performance will be for (1) year from date of award. It is anticipated that 200-300 samples will be sent for the services during the year.

The PI shall send environmental DNA amplicons to the laboratory to purify nucleotide strands, prepare barcoded libraries and sequence the libraries to provide Next Gen sequence data, as well as provide bioinformatic analysis.

II. WORK REQUIREMENTS

A. Technical Requirements.

1. The work requires:

· The development of a unique reference DNA library- including USGS-WARC data and published sequences

· Optimization of a USGS custom metabarcoding assay

· Sequencing analysis:

· Oxford Nanopore Promethion flow cell

· 2-step PCR

· High sensitivity (6 PCRs pooled)

· Super high accuracy base calling

· 100K+ reads/sample

· Read length= amplicon length

· Bioinformatic analyses

· Sequence data trimming, quality assessment and cleaning

· Generation of exact sequence variants (ESVs)

· Assignment to taxonomic IDs from reference library

· Output table with full characteristics of each ESV

· Data file of raw reads and ESVs

· Fastq files and QC report

The contractor shall be responsible for assessing, evaluating, verifying, and confirming the conditions of the samples for library preparation and next generation sequencing, followed by bioinformatic analysis.

The Contractor is responsible for receiving the samples and all molecular processing of the samples therein.

Responsibility and liability for loss or damage of samples shall remain with the contractor until final inspection and acceptance of sequences by the USGS.

2. Criteria for acceptance of Product or Service.

Results will be delivered within six weeks of sample intake by the contractor, barring unforeseen issues in processing. Contractor will include appropriate number of negative control samples, and these will have results that are to the satisfaction of the requestor.

B. Deliverables.

1. Characteristics for work product described in II.A.1., identified by name and quantity.

Deliverables are required to be transmitted securely and must adhere to QA/QC standards appropriate for publication quality. Samples will be submitted in batches. For each batch or project, the deliverables will consist of:

· 100K+ reads/sample

· Assignment to taxonomic IDs from reference library with QA/QC checks

· Output table with full characteristics of each ESV

· Data file of raw reads and ESVs

· Fastq files and QA/QC report

2. Delivery dates for work/product to be completed and point of delivery.

Services will be completed within six weeks of sample intake by the contractor. Results will be delivered electronically.

III. SUPPORTING INFORMATION

A. Place of Performance.

Contractor’s facility.

Laboratory space needs to be organized based on best practices for eDNA samples; at a minimum, separate dedicated space for pre-PCR and post-PCR processing, sterile handling of low concentration DNA samples to prevent cross contamination, etc.

B. Period of Performance

Date of Award + 365 days

C. Government Furnished Property (GFP): list property and its value.

N/A

D. Special Considerations.

1. Key personnel qualifications (researchers, instructors, writers, project managers, programmers, etc.)

The key personnel need to be experienced in the laboratory and bioinformatic analysis of eDNA samples. Personnel and analytical processes should address particular challenges associated with eDNA samples, including limitations within reference library data, difficulties related to taxonomic identification, and the potential for contamination or tag jumping.

2. Key requirements for laboratory work Laboratory space needs to be organized based on best practices for eDNA samples; at a minimum, separate dedicated space for pre-PCR and post-PCR processing, sterile handling of low concentration DNA samples to prevent cross contamination, etc.

3. Data Rights, Safety, Environment.

a. Responsibility and liability for loss or damage of samples shall remain with the contractor until final inspection and acceptance of sequences by the USGS. All sequences shall be delivered in a secure manner and be of publication quality.

b. The Contractor is responsible for offsite disposal of all chemical and biohazardous waste generated during the sample preparation and sequencing process.

3. Security/Privacy issues (ingress to Federal buildings, personnel clearances, computer security). None