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Amplicon Library Preparation and Sequencing of seawater eDNA samples for PMEL Omics Statement of Work 01 June 2023

NR-MAH000-23-00888

Period of Performance:

Start Date: 31 August 2023 End Date: 30 August 2024

Scope of work:

The Pacific Marine Environmental Laboratory (PMEL) Ocean Environment Research Division (OERD) core mission objectives include the assessment of marine biodiversity and ecosystem function. A key tool in meeting mission requirements is the genetic analysis of environmental DNA (“eDNA”) samples generating millions of DNA sequences from amplicon sequencing of filtered sea water. Such analysis is performed on a periodic basis to generate a time series by which ecosystem changes can be monitored. Therefore, procurement of sequencing services that meet the specific requirements for that particular time series generation are needed. In this case, the service requested is for massively high-throughput sequencing of eDNA to serve the time series generated from samples collected from the West Coast Ocean Acidification, Ecosystem & Fisheries-Oceanography Coordinated Investigations, and Olympic Coast National Marine Sanctuary cruises.

The PMEL Omics group is looking to secure a contract for amplicon library preparation and sequencing of PCR products derived from seawater environmental DNA (eDNA) samples. This includes library preparation (indexing/barcoding, normalization and general quality control) for up to 1,000 samples and for 5 distinct metabarcoding loci (5,000 total libraries). The samples will be pooled, sequenced and checked for coverage/depth after each run on a NovaSeq lane by the contractor. Samples will arrive in multiple batches depending on availability and international shipping.

Requirements:

To compare to existing data, the new data must be generated with the same platform and sequencing chemistry, as outlined below. Data delivery must be conducive to our bioinformatic workflow to ensure rapid and error-free processing, including demultiplexing of individual samples into fastq formatted files. The vendor must have a proven track record of success sequencing eDNA from filtered marine waters as well as a track record of working with amplicon sequencing and microbiome sequencing.

The contractor must have capabilities to handle varying quality of input DNA to perform successful amplicon sequencing and the ability to adjust the sequencing scheme (e.g., adjust relative quantities to ensure equal molar pooling and normalized sequencing depth, the ability to troubleshoot or remove problematic samples) based on the data output and standard sequencing quality scores after each run. The facility needs to maintain molecular biology best practices and ensure a sterile environment to prevent cross contamination of samples with other projects. They must communicate with the PMEL Omic group throughout the project to ensure high quality data is produced.

Complete library preparation for eDNA amplicon sequencing:

· Sample multiplexing with up to 384 samples per run

· Conduct a two step library preparation protocol that leverages unique dual indexing (i5 and i7 primers are unique to each library and not shared across samples on the same sequencing run) including clean-up, quality checking, equimolar sample pooling, and library preparation using 2 x 250 bp paired-end Illumina chemistry.

· Quality checking must confirm appropriate indexed amplicon library size and concentration prior to sequencing. Appropriate size selection steps will be implemented if primer-dimer or other off target DNA fragments are observed.

· Coordinate adapter use (preferred Illumina NexteraXT adapters) so that delivered round 1 PCR product from NOAA PMEL is compatible with contractor library preparation schema.

· Must be able to produce 100,000 reads per library (i.e., per barcode per sample).

· Data delivered directly to the PMEL technical POCs via external servers and data availability for up to 3 months following completion of sequencing

Deliverables:

· Amplicon sequencing at least 100K read depth coverage of 5,000 unique eDNA libraries.

· Data to be delivered within 90 days of receipt of the samples.

· Data will be demultiplexed by library prior to returning the data. Quality scores of sequences will be evaluated between each lane of sequencing to determine the best course of action. Data delivered directly to the PMEL technical POCs via external servers and data availability for up to 3 months following completion of sequencing.

· Samples to be submitted on an ongoing periodic basis beginning August 31st, 2023 through August 30th, 2024