Introduction

Formamide is used for forensic DNA and sequencing applications to denature double-stranded DNA fragments prior to capillary electrophoresis (CE). Effective denaturation is critical for proper strand separation and migration to achieve good resolution and accurate sizing of DNA fragments. The use of poor-quality formamide can result in increased background, peak broadening or artifact peaks that can obscure true allelic or sequence peaks. In forensic laboratories, such problems can complicate data interpretation and may lead to inconclusive or incorrect results. For sequencing applications, poor fragment resolution can negatively affect basecalling accuracy and read length. Thus, we recommend using only high-quality, deionized formamide to maximize data quality and reliability and to minimize artifacts and other potential problems.

This white paper presents the results of our evaluation demonstrating that Seq-DI™ Formamide (NimaGen, Nijmegen, The Netherlands) is compatible with Promega STR Systems, the ProDye® Terminator Sequencing System, and associated Matrix Standards when used with common capillary electrophoresis instruments, including the Spectrum CE System, Spectrum Compact CE System and Applied Biosystems® 3500xL Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA).

Materials and Methods

Formamide Handling and Storage

To evaluate compatibility with spectral calibration, STR analysis and DNA sequencing, three lots of Seq-DI™ Formamide (NimaGen, Cat.# SDF-025) were tested in parallel with a reference competitor formamide commonly used in forensic and sequencing workflows. Immediately upon arrival, Seq-DI™ Formamide was dispensed into 1.5 ml polypropylene tubes. An aliquot was reserved for Time 0 evaluations, and the remaining aliquots were stored at –20 °C. At each subsequent time point, an aliquot from each lot of Seq-DI™ Formamide was thawed on ice immediately before being used to make a loading cocktail for allelic ladder or amplification products prior to analysis by CE. At each time point, the competitor formamide reference sample was handled in parallel. Formamide was stored at –20 °C for one, three or six months. As Seq-DI™ Formamide performed comparably at each time point and across lots, data from the 6-month time point of one lot is reported here.

Spectral Calibration

Seq-DI™ Formamide or the competitor formamide was used to prepare a loading cocktail for spectral calibration of the Spectrum CE System and Spectrum Compact CE System using the 5-dye PowerPlex® 5C Matrix Standard (Cat.# DG4850) and 8-dye PowerPlex® 8C Matrix Standard (Cat.# DG5010). Spectral calibrations were also performed on the Applied Biosystems® 3500xL Genetic Analyzer using the PowerPlex® 5C Matrix Standard. Two spectral calibrations were performed per instrument, one with each formamide source. All spectral calibrations were performed as described in the appropriate technical manual (1–3).

STR Analysis

Loading cocktails were prepared with both formamides as described in the appropriate technical manual for the PowerPlex® Y23 (Cat.# DC2305, DC2320), PowerPlex® ESI 17 Fast (Cat.# DC1721, DC1720) and PowerPlex® 35GY (Cat.# DC3520) Systems (4–9). For the PowerPlex® Y23 System, Stabilizer Reagent (Cat.# DM6571) was also utilized within the loading cocktail (4,7). Loading cocktails were heat denatured as directed in the applicable technical manual prior to electrophoresis on the Spectrum CE System and Spectrum Compact CE System.

Triplicate injections of PowerPlex® Y23, PowerPlex® ESI 17 Fast and PowerPlex® 35GY Allelic Ladders were performed on the Spectrum CE System (Cat.# CE1008) and Spectrum Compact CE System (Cat.# CE1304) to assess sizing precision, expressed as the maximum standard deviation of allelic ladder fragment sizes. To assess resolution, the PowerPlex® ESI 17 Fast Allelic Ladder was used. This ladder includes DNA fragments that differ by a single nucleotide (nt) as follows: D2S441 has 11.3 and 12 alleles, and D12S391 has 17.3 and 18 as well as 18.3 and 19 alleles. Data were analyzed using GeneMarker®HID Software for Spectrum CE Systems (available as local, network, and client, Cat. # CE3001, CE3010, CE3011, version 3.2.0; GMHID-Spectrum) as described in the technical manual (10). An analytical threshold of 100 relative fluorescence units (RFU) was used for the PowerPlex® Y23 and PowerPlex® ESI 17 Fast Systems, and 150 RFU for the PowerPlex® 35GY System with data generated using both sources of formamide.

To assess peak height, morphology, and signal-to-noise, 2800M Control human genomic DNA was amplified with three PowerPlex® systems. Pooled amplification reactions for the PowerPlex® Y23 and ESI 17 Fast Systems (both at 0.5 ng DNA/25 μL amplification reaction) and the PowerPlex® 35GY System (1 ng DNA / 25 μL amplification reaction) were used throughout the study as described in the appropriate technical manual (4–9). These were injected in triplicate on the Spectrum CE System and Spectrum Compact CE System using loading cocktails made with both sources of formamide.

As the Spectrum CE System can hold four plates, allowing continuous CE runs approaching two days in duration, on-deck stability of STR amplification products in the presence of loading cocktail made with either formamide was assessed. Plates containing loading cocktails with amplification products and allelic ladders were left on the deck of the Spectrum CE System after initial injections and re-injected after 24, 48 and 72 hours.

To assess sensitivity of STR amplification product detection, two sets of PowerPlex® 35GY System amplifications targeting 1 ng and 62.5 pg of human genomic DNA were performed. Amplification products were denatured in loading cocktails containing each formamide prior to CE analysis. The data were analyzed to confirm that full profiles were present with both DNA inputs. The results were expressed quantitatively in terms of allelic peak heights for each locus.

Sanger Sequencing

ProDye® Terminator Sequencing System (Cat.# CR4324, CR4302, CR4310) was used to assess performance of Seq-DI™ Formamide for sequencing applications using the positive control template and sequencing primer included in the kit [pGEM®-3Zf(+) Vector and pUC/M13 Forward Primer (24mer)]. Template DNA was sequenced, and the resulting sequencing reactions were purified as described in the technical manual (11) before being resuspended in either formamide and analyzed in triplicate using the Spectrum Compact CE System. Performance was assessed by comparing read quality metrics including trace scores, contiguous read lengths (CRL) and QV20+ scores.

Results

Spectral Calibration

Spectral calibrations using the PowerPlex® 5C and PowerPlex® 8C Matrix Standards were performed on the Spectrum CE and Spectrum Compact CE Systems. Spectral calibration using the PowerPlex® 5C Matrix Standard was performed on the Applied Biosystems® 3500xL Genetic Analyzer. All spectral calibrations passed with comparable values with both sources of formamide on all three CE systems (Tables 1, 2 and 3). Representative multi-dye emission spectra from one capillary on the Spectrum CE System for both the PowerPlex® 5C and PowerPlex® 8C Matrix Standards are shown in Figure 1, Panels A and B, respectively. Representative electropherograms from the Applied Biosystems 3500xL Genetic Analyzer for a passing spectral calibration using PowerPlex® 5C Matrix Standard denatured in each formamide are shown in Figure 1, Panel C.