Resizing reaction volumes for the ForenSeq™ DNA Signature Prep kit library preparation

Abstract

The introduction of next generation sequencing (NGS; also known as massively parallel sequencing) technology in the field of forensic genetics has been welcomed by the scientific community, above all because it complements the weaknesses of capillary electrophoresis (CE) in the analysis of genetic markers, such as single nucleotide polymorphism (SNP) typing. However, one of the main obstacles to its adoption does not seem to be the cost of the instrumentation, but rather the cost of the NGS library preparation kits. With the aim of reducing the cost of library preparation without compromising the quality of the results, we tried to scale down reaction volumes for the first two polymerase chain reactions in the amplification and enrichment phases of the targeted loci of library preparation using the ForenSeq™ DNA Signature Prep kit. We used 1 µL templated DNA input to a concentration of 1 ng/µL, instead of the 5 µL at 0.2 ng/µL recommended by the manufacturer. Our findings indicate that reduction of the library preparation volume using the ForenSeq™ DNA Signature Prep kit did not interfere with the quality and reproducibility of the DNA profiles obtained and can help lower the overall cost of NGS.

Validation of reduced volume VeriFiler™ Express PCR Amplification Kit for buccal swab samples extracted using Prep‐n‐Go™ Buffer

“…Overall, reducing the PCR volume reduces cost [4] and has been found to increase the sensitivity and reduce the amount of template DNA required [1]. It has been found that the volume can be reduced by half without impacting the quality of the profile produced [5].…”

“…The validation of reduced volume PCR has been achieved with many common multiplex STR kits, for example, the PowerPlex® 16 System [1], AmpFLSTR™ Identifiler™ PCR Amplification Kit [2], and GlobalFiler™ PCR Amplification Kit [3]. Overall, reducing the PCR volume reduces cost [4] and has been found to increase the sensitivity and reduce the amount of template DNA required [1]. It has been found that the volume can be reduced by half without impacting the quality of the profile produced [5].…”

Recent advances in forensic biology and forensic DNA typing: INTERPOL review 2019–2022

“…Validation studies have been described with the ForenSeq DNA Signature Prep kit and the MiSeq FGx system [ [338] , [339] , [340] ], with the Verogen ForenSeq Primer Mix B for phenotyping and biogeographical ancestry predictions [ 341 , 342 ], and for resizing reaction volumes with the ForenSeq DNA Signature Prep kit library preparation [ 343 ]. MPS sequence data showed excellent allele concordance with CE results for 31 autosomal STRs in the Precision ID GlobalFiler NGS STR Panel from 496 Spanish individuals [ 344 ] and from 22 autosomal STR loci in the PowerSeq 46GY panel with 247 Austrians [ 345 ].…”