Specifications_from_OLE_(FINAL).docx

NEXT GENERATION DNA SEQUENCING INSTRUMENT

SPECIFICATIONS

The Genetics Section of the National Fish and Wildlife Forensics Laboratory (NFWFL) is purchasing a new high-throughput Next Generation DNA sequencing instrument to assist with forensic analyses and research support that will complement our existing Next Generation sequencing platform. Our requirements for instrument specification are defined by compatibility with existing protocols and reagents, error rates, throughput, forensic needs, widespread support community, IT support, and per sample cost. We are looking for a machine that will run efficiently with high throughput, address a variety of forensic needs from SNP discovery for non-human species identification, characterization of individual non-human genetic markers, and de novo/re-sequencing for non-human species identification, but not be tied specifically to kits and processes for human and bacterial genome identification or cancer research.

MINIMUM REQUIREMENTS:

· COMPATIBILITY.

We require a sequencing system that performs “sequencing by synthesis” with a cyclic reversible termination approach and uses the same chemistry, library preparation kits and analysis platforms currently used for research support and forensic case work in the Genetics Section of the National Fish and Wildlife Forensic Laboratory. Validation for casework and involves extensive training and testing of the instrument. To perform a similar validation on a different NGS platform would require extensive analyst time and effort.

· LOW ERROR RATES

Both precision and accuracy are essential for the validation of new genetic markers to support forensic genetic protocols used in case work that may be presented in a court of law. Subsequently, sequencing errors and reduced coverage results in increased cost per run and more runs per project to ensure data quality. We require a sequencing system that has true (not predicted) error rate < 1% (with a low indel error rate).

· HIGH THROUGHPUT

We need a flexible high throughput platform that can perform a wide array of applications from genome sequencing to high throughput targeted amplicon sequencing. A high throughput platform is advantageous because it ultimately reduces the cost per Gigabase (Gb). Gb, or one billion nucleotides, required for a given application can vary by size of the genome (e.g. human genome versus a microbe). We require a sequencing platform that has the ability to sequence large complex non-human genomes (> 3 Gb) and/or perform large scale amplicon sequencing studies in a single run. Therefore, the platform must be able to generate at least 100 Gb of data with > 150 million reads per run to ensure proper coverage and reduce error.

· ABILITY TO PERFORM PAIRED-END READS

We are looking for a system that enables us to perform characterization of individual non-human genetic markers, and de novo/re-sequencing for non-human species identification. The majority of the species used in research support and forensic case work do not have complete reference genome sequences available, thus paired-end sequencing is required. Paired-end sequencing facilitates sequencing from both ends of a fragment and generates high quality sequences that can be aligned easily. In addition, paired-end sequencing detects genomic rearrangements and repetitive elements, two sources of potential error. Thus, we require a system that uses a standard protocol of pair-end sequencing with no additional chemistry.

· SHORT READ SEQUENCING

Much of our future work will include running single nucleotide polymorphism (SNP) maker panels. SNP workflow will involve running small to large SNP panels on hundreds to thousands of samples. We require an approach that focuses on short read lengths that greatly increase the number of loci and sample size per run. For those applications that require longer reads we currently have a sequencing system supports longer reads.

· COMMUNITY SUPPORT

Much of the work we will be performing will be novel SNP development and targeted sequencing using a wide range of DNA template qualities. Therefore, we require a large user community for trouble shooting. The platform should be older than a year (2-5 years preferred) to have an established, continuing, and active external user base that may provide additional insight into problems that arise from library development to downstream analyses.

· BENCH TOP FOOTPRINT

Due to lab space configuration, the sequencing system must be a lab bench top system.

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