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Alamut 2

Manufactured by Sophia Genetics
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Alamut 2.0 is a software tool designed for the analysis and interpretation of genetic variants. It provides a comprehensive platform for visualizing and annotating genetic data, including support for various file formats and integration with external databases.

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Lab products found in correlation

Hotstartaq master mix, by Qiagen (3 mentions) Sequencher v5, by Gene Codes (2 mentions) Sequencher v4, by Gene Codes (1 mentions) Twist human core exome enrichment system, by Twist Bioscience (1 mentions) Hiseq 4000, by Illumina (1 mentions) Seqscape v2, by Thermo Fisher Scientific (1 mentions) Bigdye terminator v3.1 sequencing kit, by Thermo Fisher Scientific (1 mentions) Multiscreen plate, by Merck Group (1 mentions) Gotaq dna polymerase, by Promega (1 mentions) Sequencher 5, by Gene Codes (1 mentions)

9 protocols using alamut 2

1

Sanger Sequencing of Putative Variant

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For the SMS variant, Qiagen HotStarTaq master mix (Qiagen, Valencia, CA) was used to amplify the putative variant and 200 flanking nucleotides using the primers 5′-TGTGGCTTTCTTTTGCACAC-3′ and 5′-TGCATCTCAAAAACCAGCAG-3′. Unincorporated primers and nucleotides were removed using ExoSAP-IT reagent (USB, Cleveland, OH, USA). Sanger capillary sequencing was used to sequence the PCR products (Macrogen, Rockville, MD), and the sequences were aligned and analyzed using Sequencher v.4.10.1 (Gene Codes, Ann Arbor, MI, USA). Mutation interpretation was conducted using Alamut 2.0 (Interactive Biosoftware, San Diego, CA, USA).
Albert J.S., Bhattacharyya N., Wolfe L.A., Bone W.P., Maduro V., Accardi J., Adams D.R., Schwartz C.E., Norris J., Wood T., Gafni R.I., Collins M.T., Tosi L.L., Markello T.C., Gahl W.A, & Boerkoel C.F. (2015). Impaired osteoblast and osteoclast function characterize the osteoporosis of Snyder - Robinson syndrome. Orphanet Journal of Rare Diseases, 10, 27.
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2

Exonic Variant Protein Impact Prediction

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Exonic variants were evaluated by widely used programs for prediction of possible interference with the function, structure or stability of a protein (Supplementary Table 5): Mutation Taster (http://www.mutationtaster.org; Ensembl transcript ENST00000336273, NM_178812; GRCh37/ Ensembl 69), SIFT and GVGD as a part of commercial Alamut 2.0 (Interactive Biosoftware, Roven, France), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/; UniProt peptide Q86UE4), PROVEAN (http://provean.jcvi.org/index.php; Human GRCh37/Ensemble 66) and, MUpro (http://mupro.proteomics.ics.uci.edu).
Gnosa S., Ticha I., Haapaniemi S, & Sun X.F. (2016). MTDH genetic variants in colorectal cancer patients. Scientific Reports, 6, 23163.
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3

Sanger Sequencing of Genetic Variants

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The primer pairs RAI1-F-5′-CCAGGGCTGTTTGAAGACC -3′ and RAI1-R-5′-AAGTCGGCGGTGGAACAG -3′, PCK1-F-5′-CTCTGCAGAAATGCCTCCTC -3′ and PCK1-R-5′-CACCTTTCTCCACCAACTCC -3′ and GRIN2B-F-5′-AATCCCACAGCTCAAAAGCA -3′ and GRIN2B-R-5′-ATGTTGGCACTTGTTGGCTT -3′ were used to amplify respectively from genomic DNA the putative variants NM_030665:c.2273G>A, NM_000834.3:c.1238A>G, NM_002591.3:c.134T>C and 200 flanking nucleotides. PCR amplification was performed using Qiagen HotStarTaq master mix (Qiagen, Valencia, CA). The following conditions were used for amplification: 1 cycle of 95°C for 5 min, followed by 40 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 30 s, and a final extension at 72°C for 5 min. Unincorporated primers and nucleotides were removed using ExoSAP-IT reagent (USB, Cleveland, OH, USA).
Sanger sequencing of the PCR products was performed by Macrogen (Rockville, MD). The sequences were aligned and analyzed using Sequencher v.5.0.1 (Gene Codes, Ann Arbor, MI, USA). Mutation interpretation analysis was conducted using Alamut 2.0 (Interactive Biosoftware, San Diego, CA, USA).
Adams D.R., Yuan H., Holyoak T., Arajs K.H., Hakimi P., Markello T.C., Wolfe L.A., Vilboux T., Burton B.K., Fajardo K.F., Grahame G., Holloman C., Sincan M., Smith A.C., Wells G.A., Huang Y., Vega H., Snyder J.P., Golas G.A., Tifft C.J., Boerkoel C.F., Hanson R.W., Traynelis S.F., Kerr D.S, & Gahl W.A. (2014). Three Rare Diseases in One Sib Pair: RAI1, PCK1, GRIN2B Mutations Associated with Smith-Magenis Syndrome, Cytosolic PEPCK Deficiency and NMDA Receptor Glutamate Insensitivity. Molecular genetics and metabolism, 113(3), 161-170.
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4

Comprehensive Variant Annotation Pipeline

Cited 1 time
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Mutation interpretation analysis was conducted using Alamut 2.0 (Interactive Biosoftware, San Diego, CA, United States). The significance of identified alterations was screened with HGMD2, dbSNP3, and ClinVar4 database. The novel alterations were established according to the American College of Medical Genetics and Genomics (ACMG) guidelines (Richards et al., 2015 (link)).
The results were analyzed with in silico prediction software Polyphen2 (Adzhubei et al., 2010 (link)) (Polymorphism Phenotyping version 2)5, SIFT (‘‘Sorting Tolerant From Intolerant’’)6 (Kumar et al., 2009 (link)), and Mutation Taster7 (Schwarz et al., 2014 (link)).
Toth-Bencsik R., Balicza P., Varga E.T., Lengyel A., Rudas G., Gal A, & Molnar M.J. (2021). New Insights of Phospholipase A2 Associated Neurodegeneration Phenotype Based on the Long-Term Follow-Up of a Large Hungarian Family. Frontiers in Genetics, 12, 628904.
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5

Amplification and Sanger Sequencing of Genomic Regions

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The primer pairs used for the amplification of the regions of genomic DNA around the mutations are listed in online supplementary table S-4. PCR amplification was performed using Qiagen HotStarTaq master mix (Qiagen, Valencia, California, USA). The following conditions were used for amplification: 1 cycle of 95°C for 5 min, followed by 39 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 30 s, and a final extension at 72°C for 5 min. Unincorporated primers and nucleotides were removed using ExoSAP-IT reagent (USB, Cleveland, Ohio, USA). Sanger dideoxy sequencing of the PCR products was performed by Macrogen (Rockville, Maryland, USA). The sequences were aligned and analysed using Sequencher V.5.0.1 (Gene Codes, Ann Arbor, Michigan, USA). Mutation interpretation analysis was conducted using Alamut 2.0 (Interactive Biosoftware, San Diego, California, USA).
Davids M., Kane M.S., He M., Wolfe L.A., Li X., Raihan M.A., Chao K.R., Bone W.P., Boerkoel C.F., Gahl W.A, & Toro C. (2015). Disruption of Golgi morphology and altered protein glycosylation in PLA2G6-associated neurodegeneration. Journal of medical genetics, 53(3), 180-189.
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6

Exome Sequencing and TYMP Variant Annotation

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Library preparation, exome capture, sequencing, and variant annotation were performed by IntegraGen SA (Evry, France). Genomic DNA was captured using the Twist Human Core Exome Enrichment System (Twist Bioscience, OR, USA) and IntegraGen Custom, followed by paired-end 75 bases massively parallel sequencing on Illumina HiSeq4000. Analysis of exome data was performed using Sirius software (IntegraGen SA). TYMP variants were described based on the longest isoform (NM_001953.4) using Alamut 2.11 (Sophia Genetics, Lausanne, Switzerland) and Human Genome Variation Society guidelines.
Gautheron J., Lima L., Akinci B., Zammouri J., Auclair M., Ucar S.K., Ozen S., Altay C., Bax B.E., Nemazanyy I., Lenoir V., Prip-Buus C., Acquaviva-Bourdain C., Lascols O., Fève B., Vigouroux C., Noel E, & Jéru I. (2022). Loss of thymidine phosphorylase activity disrupts adipocyte differentiation and induces insulin-resistant lipoatrophic diabetes. BMC Medicine, 20, 95.
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7

Sanger Sequencing for LMNA Variants

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LMNA variants were confirmed by Sanger sequencing with the BigDye Terminator v3.1 sequencing kit (Thermo Fisher Scientific, Waltham, MA, USA) after polymerase chain reaction (PCR) amplification of exon 9 and flanking intronic sequences. Data were analyzed on a 3500xL Dx device with the SeqScape v2.7 software (Thermo Fisher Scientific, Waltham, MA, USA). LMNA variants were described based on the longest isoform (NM_170707.2) using Alamut 2.11 (Sophia Genetics, Lausanne, Switzerland) and Human Genome Variation Society guidelines.
Jéru I., Nabil A., El-Makkawy G., Lascols O., Vigouroux C, & Abdalla E. (2021). Two Decades after Mandibuloacral Dysplasia Discovery: Additional Cases and Comprehensive View of Disease Characteristics. Genes, 12(10), 1508.
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8

Genetic Screening for C2 Deficiency

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Genetic testing was performed on 212/273 patients (77.7%). Genomic DNA was extracted and purified from whole blood using standard procedures. Screening of type 1 C2 deficiency was performed by polymerase chain reaction (PCR) and gel migration on agarose as previously described (29 (link)). Amplifications by PCR (GoTaq DNA Polymerase, Promega, USA) of all exons and flanking splices sites of all the complement components were performed using specific forward and reverse primers (available on request). PCR products were purified (Multiscreen plates, Millipore, USA) and sequenced by the Big Dye terminator cycle sequencing methods using the same forward and reverse primers that for amplification (QiaQuick PCR Purification Kit, Qiagen SA, Hilden, Germany). Sequence analyses were then conducted (Applied Biosystems, Waltham, Massachusetts, USA) and read using Sequencher 5.0 (Gene Codes Corporation, USA). The interpretation of mutations was performed by comparing sequences of patients with the reference sequences exons using Alamut 2.3 (Interactive Biosoftware, France).
El Sissy C., Rosain J., Vieira-Martins P., Bordereau P., Gruber A., Devriese M., de Pontual L., Taha M.K., Fieschi C., Picard C, & Frémeaux-Bacchi V. (2019). Clinical and Genetic Spectrum of a Large Cohort With Total and Sub-total Complement Deficiencies. Frontiers in Immunology, 10, 1936.
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9

Predicting Splicing Changes via Computational Tools

Cited 1 time
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We used Alamut 2.3 (Interactive Biosoftware, Rouen, France), which combines the results of 7 splicing prediction algorithms— Human Splicing Finder (www.umd.be/HSF),29 (link) GeneSplicer (http://www.cbcb.umd.edu/software/GeneSplicer),30 (link) MaxEntScan (genes.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html),31 (link) NNSplice,32 (link) Splice Site Finder-Like,33 ESE-Finder (http://rulai.cshl.edu/tools/ESE), and RESCUE-ESE (http://genes.mit.edu/burgelab/rescue-ese/)—to predict whether a synonymous variant would alter predicted intron–exon splicing patterns. We used the Genome Variation Server (http://gvs.gs.washington.edu/GVS137/index.jsp) to determine whether the common (MAF ≥0.01) synonymous variants were in linkage disequilibrium.
Wambach J.A., Wegner D.J., Heins H.B., Druley T.E., Mitra R.D., Hamvas A, & Cole F.S. (2014). Synonymous ABCA3 Variants Do Not Increase Risk for Neonatal Respiratory Distress Syndrome. The Journal of pediatrics, 164(6), 1316-1321.e3.
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