A QIAamp DNA Extraction Kit (QIAGEN, Hilden, Germany) was used to extract genomic DNA from whole blood from each individual, according to the manufacturer's instructions. We stored the extracted DNA samples at −20°C until analysis. PCR was conducted using a Swift Maxi thermal cycler (Esco Micro Pte. Ltd., Singapore), followed by Sanger sequencing analysis to identify sequence variants in all 17 exons of the SEMA3D gene in HSCR patients using BigDye Terminator V3.1 Cycle Sequencing Kits (Applied Biosystems, Foster City, CA) and a 3730xl Genetic Analyzer (Applied Biosystems), with DNA Sequencing Analysis Software (Applied Biosystems) 0.1 (7 (link)). The primer sequences for SEMA3D rare variant analysis were chosen based on a previous study (4 (link)).
Dna sequencing analysis software
Manufactured by Thermo Fisher Scientific
Sourced in United States
DNA Sequencing Analysis Software is a bioinformatics tool that enables the processing, visualization, and analysis of DNA sequence data. It provides functionalities for sequence alignment, variant calling, and data annotation.
Automatically generated - may contain errors
Lab products found in correlation
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (3 mentions)
3730xl genetic analyzer, by Thermo Fisher Scientific (2 mentions)
Abi 3130 genetic analyzer, by Thermo Fisher Scientific (2 mentions)
Bigdye seq kit v3, by Thermo Fisher Scientific (2 mentions)
Qiaamp dna extraction kit, by Qiagen (1 mentions)
Veriti 96 well thermal cycler, by Thermo Fisher Scientific (1 mentions)
3730xl dna analyser, by Thermo Fisher Scientific (1 mentions)
Hotstartaq plus master mix kit, by Qiagen (1 mentions)
5 protocols using dna sequencing analysis software
1
SEMA3D Gene Variant Analysis in HSCR
2
Sequencing TP53 Exons 4-8 for Mutations
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Exons 4 – 8 of TP53 were sequenced in search of mutations. The primers used for the PCR amplification of TP53 DNA and cDNA sequences and sequencing primers are listed in tables A1 and A2 (Additional file
1 ). TP53 sequencing was performed using BigDye Seq kit v3.1 (Applied Biosystems, Foster City, CA, USA). The sequences were analyzed with the ABI 3130 genetic analyzer and DNA Sequencing Analysis Software (Applied Biosystems).
3
Screening NRG1 Exons for Mutations
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To identify mutations, all 16 exons of the NRG1 that had been PCR amplified in 20 fragments were screened using Sanger sequencing analysis with a BigDye Terminator V3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). The products were separated and analyzed on a 3730xl Genetic Analyzer (Applied Biosystems, Foster City, CA) using DNA Sequencing Analysis Software (Applied Biosystems, Foster City, CA) [17 (link), 18 (link)].
4
TP53 Gene Mutation Analysis
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The TP53 gene mutations were analyzed in exons 5 to 8. The primers used for the PCR amplification of cDNA sequences and sequencing primers are listed in table S1 . cDNA sequencing was performed using BigDye Seq kit v3.1 (Applied Biosystems, Foster City, CA, USA). The sequences were analyzed with the ABI 3130 genetic analyzer and DNA Sequencing Analysis Software (Applied Biosystems, Foster City, CA, USA).
5
Mitochondrial DNA Sequencing Protocol
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HV1 (15,971 -16,410 rCRS) and HV2 regions (15 -389 rCRS) of the reference and 240 minute heat-treated samples (n=10) were sequenced by CE systems for concordant data.
PCR amplification was prepared with the HotStarTaq Plus Master Mix Kit (Qiagen, Hilden, Germany) using 4 ng genomic DNA and 0.5 µM HV1 and HV2 M13-tailed primers [31, 32] .
The Veriti ® 96-Well Thermal Cycler (Applied Biosystems, CA, USA) was used for amplification with parameters set to 95°C for 5 minutes followed by 35 cycles of 94°C for 30 seconds, 62°C for 45 seconds, 72°C for 1 minute and final extension of 72°C for 10 minutes [33] . Products were sequenced using the BigDye™ Terminator v3.1 Cycle Sequencing Kit on the 3730XL DNA Analyser (Applied Biosystems, CA, USA) [33] . Raw DNA sequences were analysed and aligned using DNA Sequencing Analysis Software (Applied Biosystems, California, USA).
PCR amplification was prepared with the HotStarTaq Plus Master Mix Kit (Qiagen, Hilden, Germany) using 4 ng genomic DNA and 0.5 µM HV1 and HV2 M13-tailed primers [31, 32] .
The Veriti ® 96-Well Thermal Cycler (Applied Biosystems, CA, USA) was used for amplification with parameters set to 95°C for 5 minutes followed by 35 cycles of 94°C for 30 seconds, 62°C for 45 seconds, 72°C for 1 minute and final extension of 72°C for 10 minutes [33] . Products were sequenced using the BigDye™ Terminator v3.1 Cycle Sequencing Kit on the 3730XL DNA Analyser (Applied Biosystems, CA, USA) [33] . Raw DNA sequences were analysed and aligned using DNA Sequencing Analysis Software (Applied Biosystems, California, USA).
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