PCR (20 ng of DNA) and LT-RPA (5 ng of DNA, 10.5 mM of Mg(OAc)2 and 40 min incubation at 32°C) amplicons of HT17, NR24, CAT25, BAT26, D2S123, D18S61, D12ATA63, REN and HPRTII microsatellites (shorter primers were used for REN and HPRTII and are indicated in Supplementary Table S1 ) obtained for each blood sample were purified using Beckman Coulter™ Agencourt AMPure XP beads (Thermo Fischer Scientific), quantified and pooled in equimolar ratio. 900 ng of each pool were used for the ligation of dual-indexed adapters using QIAseq 1-Step Amplicon Library Kit (Qiagen) and no supplemental PCR amplification of the libraries was required. Libraries were assessed for quality and quantity with a Fragment Analyzer (Agilent) and QIAseq™ Library Quant Assay Kit (Qiagen) respectively. 50 pM of the pooled libraries were deposited on an iSeq 100 cartridge (Illumina) together with 20% of 50 pM PhiX control v3 Library (Illumina). Amplicon sequencing was performed on an iSeq 100 using 151 cycles of paired-end sequencing. FastQ files were generated for each sample using Local Run Manager Software (Illumina) and the read counts and sequences of each microsatellite allele were obtained from the aligned reads using an in-house developed Python code.
Iseq100 cartridge
Manufactured by Illumina
Sourced in United States
The ISeq100 cartridge is a consumable component used with the ISeq100 System, a benchtop next-generation sequencing (NGS) platform developed by Illumina. The cartridge is responsible for housing and processing the samples during the sequencing run, providing the necessary reagents and fluidics required for the sequencing process.
Automatically generated - may contain errors
Lab products found in correlation
Fragment analyzer, by Agilent Technologies (1 mentions)
Phix control v3 library, by Illumina (1 mentions)
Qiaseq library quant assay kit, by Qiagen (1 mentions)
Qiaseq 1 step amplicon library kit, by Qiagen (1 mentions)
Local run manager software, by Illumina (1 mentions)
Iseq 100 sequencing system, by Illumina (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
3 protocols using iseq100 cartridge
1
Microsatellite Amplicon Sequencing Protocol
2
Illumina iSeq100 Paired-End Sequencing
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The library was loaded into a 300-cycle iSeq100 cartridge (Illumina; San Diego, CA, USA) with an ISeq100 sequencing system (Illumina; San Diego, CA, USA). The following parameters were used for sequencing:
GenerateFastq mode
Library Prep Kit: Nimagen IDX96-U01
Read type: paired-end
Read lengths: read1 = 151, index1 = 10, index2 = 10, read2 = 151
Adapter trimming on
GenerateFastq mode
Library Prep Kit: Nimagen IDX96-U01
Read type: paired-end
Read lengths: read1 = 151, index1 = 10, index2 = 10, read2 = 151
Adapter trimming on
3
Targeted cDNA Amplification and Sequencing
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1 µl of the cDNA was then used for each pooled PCR in the following 10 µl Q5 polymerase reaction mix (NEB) (1 µl 10x Q5 buffer, 0.8 µl 10 mM dNTPs, 0.5 µl 10 µM pooled primers (Pool 1 or 2), 0.1 µl Q5 polymerase) using cycling protocols of 98°C 15s, 63°C 3 min s, 38 cycles). The two pools were combined after PCR and purified using 18 µl AmpureXP beads (Beckman-Coulter) as per manufacturer’s protocol and eluted in 20 µl water. 10ng of the amplified product was prepared for library with ligation and barcoded using the NEXTFLEX Rapid DNA Seq 2.0 kit (Perkins-Elmer) as per manufacturer’s instructions and eluted in 25 µl of water. Samples were pooled at 4-6 samples per run and diluted to 50pM before loading into the iSeq100 cartridge (150 nt read, paired end; Illumina) as per manufacturer’s instructions.
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