Title

P136 – Evaluation of ForenSeq NGS Assay Performance on the MiSeq i100

16:19

Wednesday August 19^th

Station 12

Duration: 12 minutes

10. NGS & SNPs

Scott Winter

The ForenSeq portfolio encompasses workflows enabling forensic DNA analysis by next-generation sequencing (NGS) for a variety of applications. These include casework STR profiling and database uploads (Signature Plus, MainstAY, and MainstAY SE), forensic investigative genetic genealogy (FIGG) using a panel of 10,230 SNPs for cold cases or large-scale human remains identification (Kintelligence & Kintelligence HT), analysis of externally visible characteristics either without or with biogeographical ancestry (Imagen and Imagen Geo), and mitochondrial DNA analysis (mtDNA Whole Genome and mtDNA Control Region). All of these workflows were initially designed to be sequenced on a single validated NGS platform, the MiSeq FGx.

The recently launched MiSeq i100 offers several advantages compared to the MiSeq FGx. Chief among these is faster run time, allowing any ForenSeq library pool to be sequenced in just 7 to 12 hours (assay dependent). Coupled with a Universal Analysis Software (UAS) server, sequencing runs started in the afternoon will finish overnight, with completed sample analysis results available the following morning. Additional advantages include reduced maintenance needs with fluidics contained within the consumable cartridges thereby eliminating post-run and maintenance washes, reagent storage at room temperature, and new reagent chemistry coupled with patterned flow cells that are more robust against over- or under-clustering.

Here we present an overview of data generated from various ForenSeq library types when sequenced on the MiSeq i100 compared to the MiSeq FGx. The 25M and 5M reagent kits for the MiSeq i100 offer similar, but slightly higher, read outputs than the comparable Standard and Micro flow cells for the MiSeq FGx, allowing the same or slightly higher sample multiplexing per run on the MiSeq i100.

Detection of the 10,230 SNPs in the Kintelligence assay was equivalent or better on the MiSeq i100, as were the tiled amplicon libraries generated with the mtDNA Whole Genome and mtDNA Control Region kits. For the existing ForenSeq assays containing STRs (Signature Plus, MainstAY and MainstAY SE) we noticed a consistent size bias on the MiSeq i100 that results in lower representation of loci covered by amplicons longer than about 300 bp. This size bias can be overcome with slight modifications to these assays to normalize interlocus balance for users intending to sequence on a MiSeq i100.

Authors

Scott Winter (QIAGEN, United States of America)
Sarah Giessner (QIAGEN, United States of America)
Quentin Gauthier (QIAGEN, United States of America)
Sarah Radecke (QIAGEN, United States of America)
Cassidy Robinhold (QIAGEN, United States of America)
Melissa Kotkin (QIAGEN, United States of America)
Kathryn Stephens (QIAGEN, United States of America)
Jonathan Shaffer (QIAGEN, United States of America)

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P136 – Evaluation of ForenSeq NGS Assay Performance on the MiSeq i100

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