Whole exome data filtering schema for DNA variants.

Genomic DNAs from five pairs of leiomyomas and corresponding myometrial samples underwent whole exome enrichment and sequencing. After sequence alignment, each pair of leiomyoma and corresponding myometrium was independently analyzed using NextGENe software (represented by each row in the table; SoftGenetics, State College, PA). The DNA variants in each tissue (leiomyoma/normal myometrium) were compared to reveal mutations which were unique to the leiomyoma. After filtering against the Single Nucleotide Polymorphism database v132 (dbSNP132; http://www.ncbi.nlm.nih.gov/projects/SNP/) and applying protein prediction software tools, PolyPhen-2 and SIFT (http://genetics.bwh.harvard.edu/pph2/; http://sift.jcvi.org/), we identified MED12 as the only gene commonly mutated in two or more leiomyomas. The minimum base coverage threshold was 20 sequencing reads per base-pair for variant filtering.aAll DNA variants in exons and within 10 base-pairs (bps) of exon-intron junctions in introns.bDNA variants unique to the leiomyoma in exons and within 10 bps of exon-intron junctions in introns.cDNA variants unique to the leiomyoma in exons and within 10 bps of exon-intron junctions in introns which were not found in dbSNP132.dExonic single nucleotide variants (SNVs) unique to the leiomyoma which caused a change in the protein sequence and were not found in dbSNP132.eSNVs unique to the leiomyoma located in introns within 10 bps of exon-intron junctions which were not found in dbSNP132.fExonic deletions/insertion-deletions (dels/indels) unique to the leiomyoma which were not found in dbSNP132.gDels/indels unique to the leiomyoma located in introns within 10 bps of exon-intron junctions which were not found in dbSNP132.hDNA variants unique to the leiomyoma in exons and within 10 bps of exon-intron junctions in introns which were predicted to be damaging to the protein, not found in dbSNP132.iLeiomyomas L2 and L3 harbored missense SNVs in exon 2 of MED12 which were detected by exome sequencing and predicted to be damaging. L1 contained a 42 bp deletion in exon 2 of MED12 which was undetected by exome sequencing but revealed via Sanger sequencing.本研究获取了5对子宫肌瘤（leiomyomas）与配对子宫肌层（myometrium）样本的基因组DNA，对其开展全外显子组富集与测序。序列比对完成后，每对子宫肌瘤与配对子宫肌层样本均使用NextGENe软件（SoftGenetics公司，宾夕法尼亚州州学院市）独立分析，对应表格中的每一行。将各组织（子宫肌瘤/正常子宫肌层）中的DNA变异进行比对，以筛选出子宫肌瘤特异性突变。通过针对单核苷酸多态性数据库v132（Single Nucleotide Polymorphism database v132，dbSNP132；http://www.ncbi.nlm.nih.gov/projects/SNP/）的过滤步骤，并结合蛋白功能预测软件PolyPhen-2与SIFT（http://genetics.bwh.harvard.edu/pph2/；http://sift.jcvi.org/）进行分析，最终确定MED12是唯一在2个及以上子宫肌瘤中发生突变的常见基因。变异过滤的最低碱基覆盖阈值为每个碱基位点20条测序读段。 a. 外显子区域及内含子中距外显子-内含子剪接位点10个碱基对（bp）范围内的DNA变异。 b. 外显子区域及内含子中距外显子-内含子剪接位点10 bp范围内的子宫肌瘤特异性DNA变异。 c. 外显子区域及内含子中距外显子-内含子剪接位点10 bp范围内的子宫肌瘤特异性DNA变异，且未在dbSNP132数据库中收录。 d. 子宫肌瘤特异性外显子单核苷酸变异（single nucleotide variants, SNVs），可导致蛋白序列改变且未在dbSNP132数据库中收录。 e. 位于内含子中、距外显子-内含子剪接位点10 bp范围内的子宫肌瘤特异性SNVs，且未在dbSNP132数据库中收录。 f. 子宫肌瘤特异性外显子缺失/插入缺失（insertion-deletions, indels）变异，且未在dbSNP132数据库中收录。 g. 位于内含子中、距外显子-内含子剪接位点10 bp范围内的子宫肌瘤特异性indels变异，且未在dbSNP132数据库中收录。 h. 外显子区域及内含子中距外显子-内含子剪接位点10 bp范围内的子宫肌瘤特异性DNA变异，经预测可损害蛋白功能且未在dbSNP132数据库中收录。 i. 子宫肌瘤L2与L3在MED12基因第2外显子携带错义SNVs，该变异经外显子组测序检出且被预测为有害变异。L1在MED12基因第2外显子携带42 bp缺失，该变异未被外显子组测序检出，但通过桑格（Sanger）测序得以确认。