Abi 3500dx genetic analyzer

Measles samples were genotyped by sequencing the N-450 region, using primers that have been previously described [12 (link)]. Sequencing reactions were performed on the Applied GenAmp 2700 Peltier Thermal Cycler (Applied Biosystems, ThermoFisher, Waltham, MA, USA) using the ABI BigDye1 Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), following the manufacturer’s protocols. Sequencing was performed on the purified amplicon template (Qia-quick PCR purification kit, Qiagen) using both forward and reverse primers. The fluorescent-labeled fragments were purified from the unincorporated terminators with the BigDye XTerminator1 Purification Kit (Applied Biosystems). The samples were injected for electrophoresis in an ABI 3500 Dx Genetic Analyzer (Applied Biosystems).

WES was carried out using an Agilent SureSelect Human All Exon V6 Kit (Agilent Technologies Inc., USA) on an Illumina NovaSeq 6000 platform (Illumina Inc., CA, USA). Data and bioinformatic analyses were performed according to a method described by a previous study (Luo et al., 2019 (link)). Candidate variants were confirmed in the parents in each family by Sanger sequencing. PCR products were bi-directionally sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) on an ABI 3500DX Genetic Analyzer (Applied Biosystems) after purification on 2% agarose gels.
Variants were described according to the nomenclature recommended by the Human Genome Variation Society (www.hgvs.org/). Variants were annotated using ANNOVAR (https://annovar.openbioinformatics.org/en/) and filtered according to their predicted effects and allele frequencies in the public database gnomAD (http://gnomad.broadinstitute.org/). Novel variants were checked in the Human Gene Variant Database (www.hgmd.cf.ac.uk) and ClinVar database (www.ncbi.nlm.nih.gov/clinvar/). InterVar (http://wintervar.wglab.org/) software was used to evaluate the pathogenicity of all variants according to the standards and guidelines of the American College of Medical Genetics and Genomics (Li and Wang, 2017 (link)).

Total DNA was isolated from FFPE tumor and paired normal tissue samples though the DNA extraction kit (TIANGEN, Beijing, China) following the manufacturer's recommendation and was used for subsequent multiplex fluorescent PCR. MSI status was assessed with the amplification of six mononucleotide repeat markers (BAT25, BAT26, NR21, NR24, MONO27, and NR 27) described either in NCI (National Cancer Institute) - or Promega- panel. In addition, the final panel also contained one gender loci (Amel) and two pentanucleotide repeat markers (Peta C and Peta D) as internal controls. Co-amplification of these targets was performed on ABI 7500 using a 25 μl reaction volume advised by MSI-testing reagent kit (SINOMDgene, Beijing, China). The PCR conditions were carried out according to the operation protocols. Fluorescent PCR products were analyzed by capillary electrophoresis using an ABI 3500DX Genetic Analyzer (Applied Biosystems) and Genemaker software 2.0 (SINOMDgene, Beijing, China).
Tumors with instability at two or more of these 6 markers were defined as MSI-H, while those without instability or showing instability at only one marker were classified as MSS and Low- Microsatellite Instability(MSI-L) tumors, respectively.

For Sanger sequencing of the 500-bp candidate regulatory region (hg38 coordinates chr1:3,774,672–3,775,171), we used the following primers: forward primer 1: 5′-TCTGTAGGGCCTCCCCAA-3′, forward primer 2: 5′-ACAGGGACCGGTCTAGCTGTA-3′, reverse primer 1: 5′-CTCCTAACAGCAGCCTCAGAG-3′ and reverse primer 2: 5′-TGTTAAGTCAGGCGACAGACC-3′. PCR products were sequenced using the BigDye Terminator v3.1 Cycle Sequencing kit on an ABI 3500 Dx Genetic Analyzer (Applied Biosystems). Additional Sanger sequencing was done at GATC Biotech, Germany. For peak calling and base determination we used CodonCode Aligner v4.1 software (CodonCode Corporation, USA).

DNA was extracted from paired tumor and normal tissues. Multiplex PCR was conducted using the Microsatellite Instability Detection Kit (Microread, China), which allowed for the detection of six markers: BAT25, BAT26, NR-21, NR-24, NR-27, and Mono27 by co-amplification using 5 μl of DNA with a concentration of 3 ng/ml. The PCR program was as follows: denaturation at 95 °C for 5 min and 40 cycles of 95 °C for 30 s, 60 °C for 1 min and 70 °C for 1 min, followed by 15 °C for 40 min. The amplicons were subjected to capillary electrophoresis on an ABI 3500 DX Genetic Analyzer (Applied Biosystems), followed by analysis using the GeneMapper v4.1 software (Applied Biosystems). If the peak for a certain marker (referred to as a left shift or right shift) was present in a tumor sample but absent in the corresponding normal sample, that marker was considered instable. MSI status was classified into three categories: MSI-H, MSI-low (MSI-L), and MSS depending on the number of instable markers (≥2, ≥1, 0).