RP-PCR for detecting GGC repeats at NOTCH2NLC (linked to NIID)8 (link), TTTCA repeats at SAMD12 (linked to benign adult familial myoclonic epilepsy)10 (link),45 (link), AAGGG repeats at RFC154 (link), and TGGAA repeats at BEAN1 (linked to SCA31)55 (link) were performed as described8 (link),10 (link),45 (link),54 (link),55 (link). RP-PCR for detecting GGCCTG repeats at NOP56 (linked to SCA36)56 (link) was performed elsewhere. Primer sequences and PCR conditions are shown in Supplementary Table 6 . RP-PCR products were resolved and visualized using an Applied Biosystems 3500xL Genetic Analyzer (Thermo Fischer Scientific) and analyzed using GeneMapper software (Thermo Fischer Scientific).
Genemapper software
Manufactured by Thermo Fisher Scientific
Sourced in United States, France, United Kingdom, Germany
GeneMapper software is a bioinformatics tool designed for DNA fragment analysis. It is used for the identification and quantification of DNA fragments generated during genetic research or forensic investigations. The software provides a platform for the analysis and interpretation of data obtained from electrophoretic separation techniques, such as capillary electrophoresis.
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Lab products found in correlation
Genescan 500 liz size standard, by Thermo Fisher Scientific (17 mentions)
3500xl genetic analyzer, by Thermo Fisher Scientific (13 mentions)
Abi 3130xl genetic analyzer, by Thermo Fisher Scientific (13 mentions)
Hi di formamide, by Thermo Fisher Scientific (13 mentions)
3130xl genetic analyzer, by Thermo Fisher Scientific (12 mentions)
Abi prism 3100 genetic analyzer, by Thermo Fisher Scientific (12 mentions)
3130 genetic analyzer, by Thermo Fisher Scientific (10 mentions)
Abi 3730 dna analyzer, by Thermo Fisher Scientific (9 mentions)
Abi prism 3130 genetic analyzer, by Thermo Fisher Scientific (8 mentions)
Abi 3730, by Thermo Fisher Scientific (8 mentions)
Multiplex pcr kit, by Qiagen (8 mentions)
Abi 3730xl dna analyzer, by Thermo Fisher Scientific (7 mentions)
Hd 400 size standards, by Thermo Fisher Scientific (7 mentions)
3500 genetic analyzer, by Thermo Fisher Scientific (6 mentions)
Abi prism 3130, by Thermo Fisher Scientific (6 mentions)
3730xl dna analyzer, by Thermo Fisher Scientific (5 mentions)
Qiaamp dna mini kit, by Qiagen (5 mentions)
3100 dna analyzer, by Thermo Fisher Scientific (5 mentions)
Md 10 linkage mapping panels, by Thermo Fisher Scientific (5 mentions)
Geneprint 10 system, by Promega (5 mentions)
389 protocols using genemapper software
1
Repeat-Primed PCR for Expanded Repeats
2
Genotyping MAO-A VNTR Variation
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The MAO-A VNTR locus was amplified using standard PCR procedures with primers as previously described.41) (link) Minor changes were implemented, including labeling the forward primer with 5′ HEX modifier, to allow for electrophoresis and visualization on a capillary sequencer. Briefly, 125 ng total genomic DNA was combined with 1× PCRx amplification buffer, 1.5 mM MgSO4, and 1×PCRx enhancer solution that accompanied the InvitrogenTM PCRx Enhancer Kit (ThermoFisher Scientific, Waltham, MA, USA), along with 0.2 mM of each dNTP, 0.0975 μg of each primer, and 0.5 U Taq polymerase in a total reaction volume of 20 μL. Cycling conditions were as previously described41) (link) with an additional denaturation step of 5 minutes at 95°C. One microlitre of the amplified product was electrophoresed and visualized using the ABI 3130 Genetic Analyzer system and GeneMapper software (ThermoFisher Scientific). Because the MAO-A gene is located on the X chromosome, males are hemizygous and females are heterozygous or homozygous at this locus. Individuals with 2, 3, or 5 copies of the MAO-A VNTR were classified as MAOA-L carriers, while those with 3.5 or 4 copies were classified as MAOA-H carriers. Female heterozygotes were categorized as MAOA-L carriers, consistent with previous research.42) (link)
3
BRCA1 Chromosome 17 STR Genotyping
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The individuals with recurrent mutations and from irrelevant families were genotyped at six different polymorphic short tandem repeats (STR) loci adjacent to BRCA1 on chromosome17, including D17S951, 17S1789, D17S846, D17S1818, D17S1327, and D17S1320. Sequences of the primers used in this study were obtained from the UCSC genome browser (http://www.genome.ucsc.edu/ ). PCR reaction was performed using fluorescently end-labeled primers with (Supplementary Table 1
4
Microsatellite Analysis of Tumor Samples
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Microsatellite analysis was performed on genomic DNA extracted from peripheral blood mononuclear cells (PBMCs) and from tumor samples, amplified by means of PCR using a carboxyfluorescein- or hexachlorofluorescein-labelled forward primer for each marker (Sigma Aldrich, St. Louis, MO, United States) and GoTaq DNA Polymerase (Promega, Fitchburg, WI, United States) following reported procedures [18 (link)]. The DNA fragments were separated by means of capillary electrophoresis (ABI 3130XL, Thermo Fisher, Waltham, MA, United States) and analyzed using Genemapper software (version 3.1, Thermo Fisher, Waltham, MA, United States). Oligonucleotide sequences are reported in Table S3 .
5
HEX-Labeled Primer PCR Amplicon Analysis
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PCR was performed as described above, but the reverse primer targeting SA2 was HEX-labelled. Fluorescent amplicons were run together with ROX1000 size standard (Asuragen, USA) under denaturing conditions in an ABI3130xl Genetic Analyzer (Thermo Fisher Scientific, USA). The results were analyzed with GeneMapper software (Thermo Fisher Scientific, USA).
6
Fluorescent SSR Genotyping Protocol
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For SSR analysis, a total of 24 SSRs designed by Ghistain et al. [36 (link)] were fluorescently labeled (6-FAM, HEX, and NED) and used for the detection of amplification products (Table S2 ). PCR reactions were carried out using 25 ul reaction mixture containing 30 ng template DNA, 1.5 mM MgCl2, 0.2 mM of each dNTPs, 0.5 um of each primer, and 1 U Taq polymerase (Inclone, Korea). The amplification was performed with the following cycling conditions: Initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 55 to 61 °C for 30 s, extension at 72 °C for 1 min, and a final extension step at 72 °C for 10 min. Each amplicon was resolved on ABI prism 3500 DNA sequence (ABI3500, Thermo Fisher Scientific Inc., Wilmington, DE, USA) and scored using a Gene Mapper Software (Version 4.0, Thermo Fisher Scientific Inc.).
7
Multiplex PCR for IG Gene Rearrangements
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For multiplex PCR, the obtained cell suspensions were centrifuged at 5000× g for 5 min. The cell pellets were resuspended in proteinase K digestion buffer (50 mM KCl, Sigma-Aldrich, St. Louis, MO, USA; 100 mM Tris-HCl pH 8.3, Sigma-Aldrich; 2.5 mM MgCl2, Sigma-Aldrich; 0.45% Nonidet P40, Boehringer, Ingelheim am Rhein, Germany; 0.45% Tween 20; Boehringer) at a proportion of 1 μL per 500–1000 cells, frozen and stored at −20 °C. The frozen cell lysates were thawed at room temperature. After thawing, proteinase K (Macherey-Nagel, Düren, Germany) was added at a concentration of 2 units per μL, and the mixture was incubated for 3 h at 56 °C; the enzyme was inactivated by heating at 95 °C for 7 min. The mixtures were vortexed for 1 min and centrifuged at 10,000× g for 5 min. The obtained supernatants were used for multiplex PCR based on 2000–4000 cells per reaction. The PCR analysis of IG gene rearrangements was based on EuroClonality/BIOMED-2 protocols [34 (link)]. All PCRs were performed in duplicate and included polyclonal (donor-derived lymphocytes) and negative controls. Fragment analysis was performed with GeneMapper software (Thermo Fisher Scientific, Waltham, MA, USA).
8
Validating LRS Splicing Events
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To validate the LRS results, the splicing events were analyzed by RT-PCR followed by Sanger sequencing and semi-quantitative quantification. Two microliters of cDNA was used as template for semi-quantitative PCR and Sanger sequencing under the conditions mentioned above. The sequences and locations of primer combinations used to amplify the obtained isoforms are listed in Table S1 and shown in Figure 1 A and Figure 2 A. For semi-quantitative PCR, HEX-labelled reverse primers were used.
For Sanger sequencing, PCR products were run on a 2% agarose gel. Bands were then excised and purified with the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Düren, Germany) and sequenced. For semi-quantitative PCR, labelled-PCR products were run together with ROX1000 (Asuragen, Austin, TX, USA) or LIZ500 (Thermo Fisher Scientific) size standards under denaturing conditions on an ABI3130xl Genetic Analyzer and subsequently analyzed with GeneMapper software (Thermo Fisher Scientific).
For Sanger sequencing, PCR products were run on a 2% agarose gel. Bands were then excised and purified with the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Düren, Germany) and sequenced. For semi-quantitative PCR, labelled-PCR products were run together with ROX1000 (Asuragen, Austin, TX, USA) or LIZ500 (Thermo Fisher Scientific) size standards under denaturing conditions on an ABI3130xl Genetic Analyzer and subsequently analyzed with GeneMapper software (Thermo Fisher Scientific).
9
Repeat expansion detection in NOTCH2NLC
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Blood samples were processed to extract genomic DNA following a standard phenol-chloroform method. We performed a repeat-primed PCR protocol to test repeat expansions in the 5′UTR of NOTCH2NLC as reported in the literature.30 (link) A saw-tooth tail pattern in the electropherogram was considered to indicate a repeat expansion. Fluorescence amplicon-length PCR was used to detect the length of GGC repeat expansions. Electrophoresis was performed on a 3500xL Genetic Analyzer (Thermo Fisher Scientific). The data were analyzed using GeneMapper software (Thermo Fisher Scientific). The length of the highest signal peak of the expanded allele was used to calculate the repeat number.31 (link) Long-read whole-genome sequencing was performed using a PromethION sequencer on 2 individuals who had a saw-tooth tail repeat-primed PCR, but no peak was seen on amplicon-length PCR. Library preparation was performed using ligation sequencing 1D kits (SQK-LSK109, Oxford Nanopore Technologies, UK) according to the manufacturer's protocol. About 800 ng DNA libraries were constructed and sequenced on the PromethION platform (Oxford Nanopore Technologies, UK).
10
Ribotyping of Bacterial Isolates
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This was based on the amplification of an intergene locus between the 16S and 23S rDNA genes. Each ribotype has a specific number of intergene locus with a different length. Amplification thus produced multiple fragments with different lengths. This fragment profile is specific for each ribotype. For ribotyping, the PCR mastermix was 25 µL Hotstar Mastermix (Qiagen, Netherlands), 0.3 µL of each primer (see Table 2 for more details), 22.4 µL nuclease free water, and 2 µL sample DNA. The cycling program was activation for 15 min at 95 °C, followed by 35 cycles of denaturation for 1 min at 94 °C, annealing for 1 min at 60 °C, and elongation for 1 min at 72 °C, with the final elongation step for 30 min at 72 °C. PCR fragments were then analyzed in an ABI310 automatic genetic analyzer (Thermo Fisher Scientific, Waltham, MA, USA) in a 50 cm capillary loaded with POP4 polymer (Thermo Fisher Scientific, Waltham, MA, USA). LIZ600 was used as the size standard (Thermo Fisher Scientific, Waltham, MA, USA). The length of each fragment was determined using the GeneMapper software (Thermo Fisher Scientific, Waltham, MA, USA). The resulting profile was then uploaded to the WEBRIBO database (https://webribo.ages.at/ ).
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