Mutations identified by WES were verified in all patients with EB and healthy family members by SS. Primers were designed on the NCBI website (National Center for Biotechnology Information, www.ncbi.nlm.nih.gov ), PCR was used for amplification, and sequencing was performed as described previously (11 (link)). All amplicons were sequenced directly on an automated DNA sequencer (3500xL Dx Genetic Analyzer; Applied Biosystems, Waltham, MA, USA).
3500xl dx genetic analyzer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 3500xL Dx Genetic Analyzer is a capillary electrophoresis instrument designed for genetic analysis. It utilizes four-color fluorescence detection to perform high-quality DNA sequencing and fragment analysis.
Automatically generated - may contain errors
Lab products found in correlation
Gel extraction kit, by Qiagen (1 mentions)
Big dye terminator reaction mix, by Thermo Fisher Scientific (1 mentions)
Bigdye xterminator kit, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator v3.1 kit, by Thermo Fisher Scientific (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Multiplex pcr plus kit, by Qiagen (1 mentions)
Genemapper v4, by Thermo Fisher Scientific (1 mentions)
Variant reporter software, by Thermo Fisher Scientific (1 mentions)
9 protocols using 3500xl dx genetic analyzer
1
Verifying Mutations Across Samples
2
MLPA for STS Gene Deletions
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We employed SALSA® MLPA® Probemix P160-C1 STS reagent (MRC-Holland, Amsterdam, The Netherlands) to assess large exon deletions in the STS gene identified through targeted panel sequencing with CNV analysis. The procedure followed the manufacturer’s protocol for multiplex ligation-dependent probe amplification (MLPA). Subsequently, capillary electrophoresis and fragment analysis were performed consecutively using a 3500XL Dx Genetic Analyzer (Applied Biosystems, Carlsbad, CA, USA). Coffalyser.Net software version 220401.000 (MRC-Holland) was utilized for the comparative analysis of CNV data in accordance with the manufacturer’s instructions. Three sex-matched normal DNA samples were employed to normalize the resulting peak intensities. Manufacturer control probes served as references for the analysis. Generally, probe-to-peak ratios ranging from 0.7 to 1.3 were considered indicative of a normal copy number (wild-type). Conversely, a loss of one copy number was defined by probe-to-peak ratios between 0.4 and 0.7. A gain of one copy number was inferred from probe results for probe-to-peak ratios between 1.3 and 1.6.
3
MPXV Sequence Confirmation via PCR
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The PCR products were purified using a gel extraction kit (Qiagen). Next, sequence reactions using the F3L, A39R, 1995MPV, 1997MPV, and HAOUT primers with the BigDye Terminator Reaction Mix (Applied Biosystems, Foster City, CA, USA) were performed. The reaction products were purified using a BigDye Purification Kit (Applied Biosystems), and sequence analysis was performed with a 3500xL Dx Genetic Analyzer (Applied Biosystems) [11 (link)]. A nucleotide blast was carried out with the sequencing results to confirm whether the sequences matched those of MPXV.
4
HIV Genotyping using Thermo Fisher Kit
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Thermo Fisher Scientific HIV Genotyping kit was used for the pol gene sequencing, and includes 6 overlapping primers. 2 µL of PCR product was mixed together with 18 µL of sequencing mix. The sequencing conditions were as follows: 25 cycles of 10 seconds at 96°C, 5 seconds at 50°C, and 4 minutes at 60°C and were carried out on an Applied Biosystems 3500xL DX genetic analyzer using the BigDye XTerminator kit (Applied Biosystems, Foster City, USA). The Stanford HIV Drug Resistance Database genotyping algorithm was used to identify drug resistance mutations from sequencing files that were automatically interpreted by RECall.[11 (link)] The pol sequences have been archived in the DDBJ Nucleotide Database [LC723952-LC724015].
5
Detecting Duchenne Muscular Dystrophy Gene Variants
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To detect duplications or deletions of the DMD gene in the proband (II-1 in Figure 1 ) and his daughter (III-2 in Figure 1 ), multiplex ligation-dependent probe amplification (MLPA) was conducted using a SALSA® MLPA® Probemix P035 DMD-1/2 (MRC Holland, Amsterdam, The Netherlands) according to manufacturer protocols. Capillary electrophoresis and fragment analysis were performed using a 3500xL Dx Genetic Analyzer (Applied Biosystems, Carlsbad, CA, USA). Comparative copy number variation data were estimated using Coffalyser.Net software ver. 210604.1451 (MRC Holland) according to manufacturer instructions. The generated peak signals were normalized to the manufacturer control probes and those of gender-matched normal DNA as reference. Estimating that the probe normally targets two copies, a probe to peak ratio of 0.7–1.3 was considered as a normal copy number (wild type). Further, a probe to peak ratio of 1.3–1.6 was considered to indicate a heterozygous duplication (gain of one copy number), and a probe to peak ratio of 0.4–0.7 was assumed to indicate a heterozygous deletion (loss of one copy number).
6
Microsatellite Instability Analysis
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A single multiplex polymerase chain reaction (PCR) reaction (Genetree Research, Seoul, Republic of Korea) amplified five microsatellite loci (NR27, NR21, NR24, BAT25, and BAT26), using genomic DNA extracted from GC and normal control samples. The PCR products were subsequently analyzed using capillary electrophoresis with 3500xL Dx Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). MSI status was determined based on the data obtained from the sequencer by independent board-certified laboratory geneticists. Interpretation criteria were as follows: instability at more than one locus was classified as MSI-H, instability at a single locus was classified as MSI-L, and no instability at any locus was classified as MSS.
7
Analyzing Gene Expression in Thymic Lymphoma
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Total RNA was extracted from thymic lymphoma cells using the Maxwell 16 RNA Purification kit and used for reverse transcription with Superscript III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and random hexamers. PCR was performed using primers for Ikzf1, Trp53, Pten, and Gapdh as described previously [16 (link), 31 (link)], with expression of Gapdh used as an internal control. PCR products were electrophoresed through agarose gels containing ethidium bromide and photographed using a digital imager (Amersham Imager 600; GE Healthcare, Chicago, IL, USA). PCR products were directly sequenced using the Big Dye Terminator v3.1 kit (Applied Biosystems, Foster City, CA, USA) and 3500xL Dx Genetic Analyzer (Applied Biosystems). The primers used for PCR and sequencing analysis are shown in S2 Table .
8
LOH Detection in Tumor Samples
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To detect LOH at 1p and 16q, 16 polymorphic microsatellite markers were selected based on a previous study [6 (link)]. Forward primers were labeled with four different fluorescent dyes (VIC, 6FAM, PET, or NED) (Table 1 ). Polymerase chain reaction (PCR) amplification was performed on tumor and normal tissue samples in triplicate using the Qiagen Multiplex PCR Plus Kit (Hilden, Germany) under the following conditions: 95°C for 10 minutes; five cycles of 95°C for 20 seconds, 60°C for 90 seconds (−1°C in each cycle), and 72°C for 1 minute; 30 cycles of 95°C for 20 seconds, 55°C for 90 seconds, and 72°C for 1 minute; and 60°C for 30 minutes. PCR products were examined using a 3500xL Dx Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA) and LOH was detected using GeneMapper v.4.1 software (Thermo Fisher Scientific) according to the manufacturer’s instructions.
9
Screening and Sequencing of CFTR Mutations
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The xTAG® Cystic Fibrosis 71 Kit v2 (Luminex Corp., Oosterhout, Netherlands) was used for initial screening of 71 CFTR mutations according to the manufacturers protocol, using 5 μl of extracted genomic DNA.
The 2 nd tier also included Sanger sequencing of c.3873 + 2T N C, present in 3.4% of Norwegian CF patients (O Rødningen, K Eiklid, KR Heimdal, A Blomhoff, O Storrøsten. personal communication). Primers and PCR conditions were from the VariantSEQr project [18] . Sequencing was performed using Applied Biosystems 3500 xL Dx Genetic Analyzer with analysis in Variant Reporter software (Thermo Fisher Scientific Inc, Waltham, MA, USA). The mutation nomenclature used throughout this manuscript relates to the reference sequences NM_000492.3 and NP_000483.3 or the CFTR2 database legacy names.
The 2 nd tier also included Sanger sequencing of c.3873 + 2T N C, present in 3.4% of Norwegian CF patients (O Rødningen, K Eiklid, KR Heimdal, A Blomhoff, O Storrøsten. personal communication). Primers and PCR conditions were from the VariantSEQr project [18] . Sequencing was performed using Applied Biosystems 3500 xL Dx Genetic Analyzer with analysis in Variant Reporter software (Thermo Fisher Scientific Inc, Waltham, MA, USA). The mutation nomenclature used throughout this manuscript relates to the reference sequences NM_000492.3 and NP_000483.3 or the CFTR2 database legacy names.
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