B08_140G0221R0028_Attch_1_PWS.doc

Performance-based Work STATEMENT

Sequence data from a metagenome sequencing facility

I. General Information Introduction: The Department of the Interior (DOI) U.S. Geological Survey (USGS) has a requirement for sequence data analysis services. Genomics, metagenomics, and transcriptomics allow investigators to analyze genetic material recovered directly from environmental samples. This type of analysis allows researchers to identify the complete set of DNA within a single organism of interest (genomics), reveal microbial diversity within natural samples (metagenomics) or investigate what genes are being expressed at a particular point in time (transcriptomics). Gaining this kind of insight into the microbial world, which has for years been considered a black box, is extremely useful for understanding and making predictions about changes in environmental processes.

Background: Microbial ecologists within the USGS encompass a broad spectrum of research interest that relate to understanding microbial processes as it relates to environmental health and improving our Nation’s water quality. The success of this work, and of the USGS mission, requires not only high-quality analytical results, but also results that can be compared with similar data sets, many of which have been generated previously and used in USGS scientific investigations. We have research needs that include gene target amplifications, amplicon surveys, whole genome(s) sequencing, and shotgun metagenomics. USGS researchers will be responsible for DNA extraction, fluorescence-based quantification, normalization, and gel electrophoresis for gene target verification before sending samples to the vendor.

Scope: Services, non-personal, to provide all plant equipment, supervision, labor, travel, and materials (unless otherwise provided herein) necessary to provide DNA or RNA amplification and barcoding, relative quantitation of amplicon efficiency, shotgun metagenomics, pooling of barcoded DNA, Illumina library prep, sequencing, and data file generation.

Terms and Acronyms Used in Work Statement:

DNA. Deoxyribonucleic acid (DNA) is a double stranded molecule that encodes the genetic instructions used in the development and functioning of all known living organisms and many viruses

RNA. Ribonucleic acid (RNA) is a molecule that is a single stranded molecule that encodes the genetic instructions used in the development and functioning of all known living organisms and many viruses

PCR. The polymerase chain reaction (PCR) is a biochemical technology in molecular biology used to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.

Illumina MiSeq is an instrument used for applications including small whole-genome sequencing (microbe, virus), targeted gene sequencing (amplicon-based, gene panel), targeted gene expression profiling, miRNA and small RNA analysis, DNA-Protein Interaction Analysis (ChIP-Seq), and 16S Metagenomic sequencing Illumina NextSeq is an instrument that allows for all the analysis included in Illumina MiSeq but in addition allows for the analysis of exome and large panel sequencing (enrichment based), Single-cell Profiling (scRNA-Seq, scDNA-Seq, oligo tagging assays), transcriptome sequencing (total RNA-Seq, mRNA-Seq, gene expression profiling), methylation sequencing, metagenomic profiling (shotgun metagenomics, metatranscriptomics), and cell-free sequencing and liquid biopsy analysis.

Illumina NovaSeq is an instrument that allows for whole-genome, whole-exome, and whole-transcriptome sequencing.

Sanger sequencing is a method that yields information about the identity and order of the four nucleotide bases in a segment of DNA. It provides a high degree of accuracy, long-read capabilities, (> 500 nucleotides) and the flexibility to support a diverse range of applications in many research areas. It is beneficial for the use in smaller-scale projects, and for the validation of Next-Gen results.

II. Work Requirements

a. Technical Requirements:

Task 1. Prepare DNA or RNA into sequencing-ready Illumina libraries and perform quality control assays: Qubit quantification using the dsDNA HS assay, TapeStation D1000 HS assay (to assess the size distribution of the sample and check for any free primers and/or primer-dimers), and Kapa qPCR for library quantification. If Core-prepared libraries have QC problems, they will be subjected to an additional round of clean up if primer-dimers are an issue, or the libraries will be remade. Illumina libraries must pass QC will be sequenced.

Task 2. Generated data from Illumina sequencing will be processed using Illumina's bcl2fastq software to convert data from BCL format to fastq files and demultiplexes read data into individual fastq files, as necessary, based on index (barcode) sequences associated with each library. Quality of the output data will be checked using the program FastQC and the final FastQC report file (.html) for each read of each library sequenced is returned to the submitter along with their sequence data. Illumina's performance parameters will be used to judge a run according to total output and quality. These are specific for each instrument and run format but as an example the performance specifications for the NovaSeq 6000 are shown at https://www.illumina.com/systems/sequencing-platforms/novaseq/specifications.html. Performance specifications for the MiSeq are shown at https://www.illumina.com/systems/sequencing-platforms/miseq/specifications.html. Specifications for the NextSeq can be found at https://www.illumina.com/systems/sequencing-platforms/nextseq/specifications.html. In cases of reagent problems, instrument failure or library preparation problems (for vendor prepared libraries), additional sequencing will be performed at no cost to the researcher to produce a total number of sequences that meet Illumina's standards for the requested sequencing run.

Task 3. The contractor shall produce a fastq reads archive that contains the following files: 1. Sequencing files in FASTQ format 2. A summary file consisting of sample names, barcodes, number of reads per sample, and quality scores of forward and reverse reads. 3. Method summary including relevant references. 4. Inclusion of a positive control mock community with each run.

Task 4. Unused sample will be returned to the USGS at an agreed upon price, if requested. Otherwise, the contractor will be responsible for disposing unused sample.

b. General Requirements:

1. The company must be able to fulfill all the technical requirements listed above and perform the work at a competitive price.

Deliverables:

The contractor shall deliver the fastq files, a summary file with quality scores, and a method summary. The contractor shall deliver the results electronically within 90 days from the date the sample is received.

(End of Work Statement)

QUALITY ASSURANCE SURVEILLANCE PLAN (QASP)

The contractor is responsible for quality control and must complete all quality control plan measures before turning results to the Technical Liaison for the Government’s Quality Assurance of the contractor’s performance.

The Government will include positive and negative blind samples for standard assurance with each sample batch. The contractor’s results will be compared to prior results of the blind sample and used to assure the quality standards described in the work statement are met.

If/when the results are substandard, the contractor shall reanalyze the sample batch in response to the Government’s substandard performance notice, at no added cost to the Government.