assay characteristics performance parameters fmf mastr v2 a simple and robust research assay for complete mutation analysis in the coding regions of the mefv gene by next-generation sequencing. mutations of the mefv gene affect normal functioning of the pyrin protein, leading to the inflammatory disorder familial mediterranean fever (fmf). 100% 90% 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 80% 70% 60% 50% 40% 30% 20% 10% 0% percentage of amplicons x mean coverage graph representing the read pair counts for all 23 fmf mastr v2 amplicons, showing their uniform representation. to allow comparison between samples, the read counts were normalized. application ? for the identification of mutations (snvs and indels) in the mediterranean fever gene ( mefv ). ? for complete mutational spectrum analysis in one assay ? ready-to-use kit with simple-to-follow protocols gene analyzed mefv genomic region analyzed 4.2 kb all exons 30 bp fanking region number of amplicons 23 amplicon length 250-400 bp number of plexes 2 dna amount required 20 ng per multiplex reaction compatible with miseq system, illumina fmf mastr v2 on miseq; germline dna dna extracted from blood on target read pair counts 94.6 % [87.6 % - 97.6 %] uniformity of amplifcation (0.2x mean coverage) 99.4 %
? agilent technologies, inc. 2017 printed in belgium, december 31, 2017 5991-8953enn pr7000-1436 order info for research use only. not for use in diagnostic procedures. multiplicom n.v. [ a ] galile?laan 18, b-2845 niel, belgium [ w ] www.agilent.com [ e ] mtp.customerservice@agilent.com [ t ] +32 3 289 14 00 [ f ] +32 3 400 23 16 cat. no. product name reactions mr-0071.024 fmf mastr v2 24 mid (molecular identifiers) kits are necessary to complete the workflow. advised maximum number of samples per run workflow sequencing system illumina miseq reagent kit flow cell nano v 2 2 x 251 cycles v2 2 x 251 cycles v3 2 x 276 cycles snv variant calling minimal coverage per allele: 50 116 1753 3215 plan run multiplex pcr quality control universal pcr quality control equimolar pooling mixing plexes of single sample puri? cation multiplex pcr dilution universal pcr mixing clean up pooling sequencing data analysis mastr work? ow ngs
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