The ForenSeq DNA Signature Prep Kit contains two primer mixes to target specific regions of the genome. The mixes enable analysis of autosomal Y- and X-chromosome STR targets, identity-informative SNPs, with the option to include ancestry-informative, and phenotypic-informative SNPs. Libraries are prepared from an individual DNA sample, which can be multiplexed with libraries of other individual DNA libraries for sequencing on the MiSeq FGx Instrument.
Where CE-based assays give the length of the STRs, Illumina technology provides, not only the length of the STRs, but any variation in the STRs and genotypes at the various SNPs. Illumina technology also allows multiple samples to be pooled together and sequenced in one run, instead of running samples one at a time.
Expiration dates are printed on the kit box labels and reagent tubes. Illumina tries to ensure 6 months of shelf life from time of kit shipment.
The ForenSeq DNA Signature Prep Kit is used for two primary applications: Criminal Cases and Database, but can also be used in other applications, such as Missing Persons, Mass Fatality, and Bio-geographical Ancestry.
The kit contains three boxes.
The ForenSeq DNA Signature Prep Kit is used to determine which STRs and SNP genotypes are present in an individual sample.
DNA Primer Mix A—Contains primer pairs for 59 STRs (including 27 autosomal STRs and 8 X and 24 Y haplotype markers) and 95 identity-informative SNPs.
DNA Primer Mix B—Contains all markers in DNA Primer Mix A, plus primer pairs for 56 ancestry-informative SNPs and 22 phenotypic-informative SNPs (two ancestry-informative SNPs are also used for phenotype prediction).
Preparing libraries takes approximately 9 hours. Sequencing and Real-Time Analysis (RTA) software tasks on the MiSeq FGx such as image processing, assigning base calls, and then designating a quality score for each base call takes approximately 30 hours. Aligning reads to provide sample information, along with optional steps such as generating reports, population statistics, sample comparison, phenotype estimations and bio-geographical ancestry estimations take approximately 1 hour.
The ForenSeq DNA Signature Prep Kit is for use with human DNA samples only.
A fluorometric based method, such as qPCR is recommended for quantifying gDNA.
Degraded DNA generates libraries with the kit. Depending on the extent of degradation, not all of the large amplicons in the multiplex can be detected. The kit can also overcome high levels of inhibitors that can be present in forensic samples.
Follow these DNA input recommendations:
Final libraries are single stranded and cannot be quantified. Follow the normalization and pooling protocol before loading onto the MiSeq FGx.
Contamination is always a concern with PCR. Illumina recommends setting up PCR1 in a Pre-PCR facility. Illumina recommends using two areas in the post-PCR area: one for setting up PCR2 (no libraries present in this area) and a separate area for the library purification, bead-based normalization, and MiSeq FGx set-up.
Caps are provided to prevent cross-index contamination. After a cap has been removed from a tube, it is discarded and a fresh cap is used to seal the tube after use.
The ForenSeq DNA Signature Prep Kit includes two positive controls: an assay control, which is processed with the samples through library preparation, and a sequencing control, which is a prepared library included with the pooled samples before sequencing. Two negative controls can also be included: a reagent blank, which is included from the DNA extraction step, and a PCR blank, which is a nuclease-free water sample processed through library preparation.
Processing fewer than 8 samples at one time, including positive and negative controls, can cause problems with pipetting accuracy, due to the small volumes used when preparing the master mix.
8–96 single-source database or reference samples can be processed and pooled and 8–32 casework or evidentiary samples can be processed and pooled.
A full list of user supplied items can be found in the Consumables and Equipment section of the ForenSeq DNA Signature Prep Guide or ForenSeq DNA Signature Prep consumables and equipment excerpt. Comparable performance is not guaranteed when using alternate equipment.
No, the reagent blank is made by the kit user.
Choosing which primer mix to use depends on the application and legislation in a particular country. DNA Primer Mix A can be used for casework and database applications. Due to legislation in some countries, ancestral and phenotypic data cannot be stored in the database and DNA Primer Mix B can then only be used for casework.
The human sequencing control acts to make sure that the sequencing run is performing as expected. If there is an issue with the run, the human sequencing control provides information as to whether the problem is related to the instrument or library. The human sequencing controlalso acts to allow the run to complete successfully, where degraded or low quality libraries do not have the long STR sequences required to last through 351 cycles.
No. However, a sample sheet created using IEM is compatible with the RUO mode of the MiSeq FGx Control Software. For forensic runs, the samples are defined in the ForenSeq Universal Analysis Software before acquisition through a file upload or manual entry. For details on the information in the file, see the ForenSeq Universal Analsyis Software Guide.
Introducing too much DNA onto the flow cell results in a raw cluster density over the recommended 1.4M/mm2 threshold. Overclustered flow cells can result in poor quality data, as individual clusters cannot be adequately resolved and low numbers of clusters pass filters to be used in final output. It is recommended to target < 1.4M/mm2 to ensure high-quality sequencing performance.
Final pooled libraries can be stored at -25°C to -15°C for up to 3 months.