Performance Work Statement eDNA.pdf

PERFORMANCE WORK STATEMENT

Background/Overview:

Environmental DNA (eDNA) refers to all genetic material that is recovered from environmental substrates such as water or sediment. eDNA is a by-product of metabolic processes and is derived from sources such as feces, urine, skin, hair or decomposing organisms. This preserved, but often degraded, genetic material provides a means to audit species composition and communities at a given location. Metabarcoding is a technique that allows the amplification of multiple taxonomic groups simultaneously. This can be used to generate a biodiversity audit from an environmental sample containing multiple species. When combined with high-throughput DNA sequencing technologies, eDNA can provide a wealth of information for biodiversity studies of the detection of species in a rapid, cost-effective and non-destructive manner.

Purpose & Objectives:

The Pacific Islands Fisheries Science Center (PIFSC) does not possess the equipment required to process and sequence eDNA samples. Therefore, a commercial laboratory is required to process and sequence these samples collected by PIFSC. In short, the PIFSC will provide samples so that eDNA can be extracted, amplified using a combination of universal and taxa-specific assays, and then sequenced. The combination of multiple high-throughput assays can reveal greater diversity of species and finer taxonomic resolution.

Objectives:

The PIFSC requires laboratory processing services including library preparation and sequencing. The PIFSC will collect and filter water samples using polyethersulfone membranes (PES, filters) with a vacuum pump to capture eDNA present in the water. Filters will be frozen shortly after collection and kept frozen until and during shipping to the Contractor’s location via overnight shipment on dry-ice.

The Contractor shall process and sequence each sample. Processing includes extracting DNA from water filters, quantifying DNA, PCR amplification and indexed adapter ligation, library blending and purification, Illumina sequencing, and demultiplexing raw sequencing reads.

Scope of Work:

Contractor Laboratory Workflow Overview

DNA is extracted from incoming samples by a hybrid manual/automated method using commercially supported reagents.

The Contractor shall use the metabarcoding assays noted below to target a wide diversity of marine species of relevance to this study.

(i) a nuclear rRNA 18S assay; invertebrate taxa

(ii) an assay to detect the mitochondrial cytochrome c oxidase 1 (CO1);

(iii) a fish mtDNA 16S assay.

(iv) an assay targeting the 12S rRNA known as MiFish.

Options for:

(v) an assay targeting the 23S rRNA for detection of algae.

(vi) coral ITS2 assays – for detection of hard and soft corals, worms, anemones, jellyfish.

The PIFSC anticipates between 115 to 800 eDNA water filter samples to be sequenced at 2-6 assays per sample. The anticipated average is 4 assays per sample, but may adjust between 2 and 6 assays per sample.

Analysis of sequence data

Sequence data will be demultiplexed by the Contractor and provided to the PIFSC who will analyze data against their database to identify present taxa.

Tasks:

All laboratory benchwork shall be conducted according to the Contractor’s preferred standard operations and procedures, but shall include the following processes:

(1) DNA extraction from water filters;

(2) DNA quantification

(3) Fusion tagging PCR (i.e., amplification and sequencing index adapter ligation)

(4) Minipooling and Quantification (post-PCR)

(5) Library blending and Purification

(6) Sequencing

(7) Demultiplexing (Post-sequencing bioinformatics) of raw sequencing reads then provided to PIFSC.

The Contractor must provide PIFSC the following:

• Sequencing data must be returned in FastQ format in clean (i.e., passing Illumina’s instrument quality assessment pipeline) de-multiplexed form.

• Basic facts of methods used must be disclosed, as would be expected for inclusion of sequencing data in the peer-reviewed literature (e.g., instrument/kit used).

• All libraries must be performed using the same techniques/kits, and all libraries must be sequenced on the same platform, unless negotiated with and approved by the PIFSC Point of Contact.

Period of Performance:

The period of performance is a one-year Base Period and three (3) one-year Option Periods.

Place of Performance: All laboratory work shall be performed at the Contractor’s facility. The Contractor’s facility shall be a commercial molecular laboratory with separate dedicated purpose-built clean rooms for each stage of eDNA genetic processing procedures (DNA extraction, PCR, and post-PCR processes) in order to minimize the risk of sample cross-contamination. In addition, the facility must possess dedicated and up-to-date eDNA equipment and access to an Illumina sequencer (e.g., MiSeq). The facility must be able to receive and store sensitive eDNA samples.

Government-Furnished Property (GFP): There will be no Government-Furnished Property with this Contract.

Security Requirements: There are no security requirements with this contract.

Applicable Standards:

• Assays are prepared with the use of isolated purpose-built ‘Ultra-Clean’ rooms. Massively parallel sequencing (also known as Next Generation Sequencing, or NGS) will be applied on the prepared metabarcoding assays. This innovative technique enables all amplified sample organisms from the collected sample to be represented in the assay result.

• This method is very sensitive to external contamination at all stages of the methodology. Multiple negative controls are included throughout the laboratory handling workflows. Work is done in dedicated purpose-built clean rooms and eDNA dedicated equipment under strict logistical protocols.

Special Requirements:

• The Contractor must possess Illumina sequencing equipment, equipment for quality and quantity assessment as appropriate for library preparation from DNA and sequencing, and equipment for library construction.

• Contractor shall provide personnel experienced in DNA extraction, library preparation, and sequencing.

• The Contractor must be able to process samples (including all steps for conversion of total DNA sample into sequence data) at an average rate of 100 samples/3 months or faster.

Deliverables Schedule:

Date Range Deliverable Activities Estimated Number of Samples

September 1, 2020 – August 31, 2021

Samples shipped to Contractor who will process according to applicable standards and procedures as outlined in this SOW

115 - 800

September 1, 2021 – August 31, 2022

Samples shipped to Contractor who will process according to applicable standards and procedures as outlined in this SOW

115 - 800

September 1, 2022 – August 31, 2023

Samples shipped to Contractor who will process according to applicable standards and procedures as outlined in this SOW

115 - 800

September 1, 2023 – August 31, 2024

Samples shipped to Contractor who will process according to applicable standards and procedures as outlined in this SOW

115 - 800

The Contractor shall provide the following:

1. Sequence data from each provided sample in FastQ format, in demultiplexed form, returned on average within 90 days of receipt of samples or faster, and in no case more than 6 months of sample receipt.

2. Overview of methods used in contractor format, as would be expected for inclusion of library preparation and sequence methods in the peer-reviewed literature for publications presenting the sequencing results. This includes names of the sequencing instrument and main kits used for library construction, cleaning/demultiplexing protocol, and statement of the number of samples sequenced per lane.

Acceptance Criteria:

Study deliverables shall be the provision of demultiplexed sequencing files, provided to PIFSC at the conclusion of sequencing.

Storage and Disposal:

Raw submitted samples (filters) may be fully used and destroyed by the DNA extraction procedure. Unused DNA sample and excess final library material not consumed in generation of sequence data could be archived at Contractor’s facility (for up to 5 years) or returned to PIFSC via overnight shipment on dry ice by August 31, 2024.

(End of Performance Work Statement)