Miseq fgx sequencer

The bioinformatically-identified STRs were amplified in a single multiplex using 3–10 ng of genomic DNA per bird with each unlabelled primer at a final concentration of 0.2 μM. Barcoded sequencing libraries were prepared with the TruSeq PCR-free Library Preparation kit (Illumina) and quantified using both the 2100 BioAnalyser system (Agilent) and Qubit™ fluorometer (Invitrogen) with appropriate dsDNA high sensitivity assays. Based on a 550 bp average insert size, libraries were normalised to 4 nM, diluted, and denatured to a loading concentration of 12 pM with a 1% ΦX spike-in in accordance with the manufacturer’s recommendations. Paired-end sequencing was performed using 500 cycles on an Illumina/Verogen MiSeq FGx sequencer in “research use only” (RUO) mode using the machine’s “Generate FASTQ” workflow. Paired-end reads were merged with FLASH version 1.2.11/lo and alleles were called by FDSTools ([56] (link)).

Genomic DNA of 1 ng was used for the test. Libraries were constructed using the ForenSeq™ DNA Signature Prep Kit (Illumina, CA, USA) following the manufacturer’s instructions. The NGSs were performed on the Miseq FGx™ sequencer (Illumina, CA, USA). The analysis of the sequencing data was performed using the built-in ForenSeq™ Universal Analysis Software (UAS, Verogen, San Diego, CA, USA) with its default settings. The reports given by UAS were provided with allele names, genotypes, and the corresponding read coverages. The default thresholds for each of the loci could be found in Supplementary Material 1, and the sequence data could be obtained from Supplementary Material 2. Depending on whether the allele was typed or not, the report could be downloaded by either Sample Summary Report (the results were given using the built-in IT) or Sample Details Report (the results were given using the built-in IT). In the present study, the STR profiles for the normal samples were genotyped using the built-in IT. The STR profiles for the tumor samples were compared using both thresholds, namely, NGS-IT and NGS analytical threshold (NGS-AT).