Selective identification of somatic mutations in pancreatic cancer cells through a combination of next-generation sequencing of plasma DNA using molecular barcodes and a bioinformatic variant filter.
Selective identification of somatic mutations in pancreatic cancer cells through a combination of next-generation sequencing of plasma DNA using molecular barcodes and a bioinformatic variant filter.
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DOI：
10.1371/journal.pone.0192611
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发表时间：
2018
期刊：
影响因子：
3.7
通讯作者：
Kato K
中科院分区：
文献类型：
--
作者：
Kukita Y;Ohkawa K;Takada R;Uehara H;Katayama K;Kato K
用于检测血浆DNA中肿瘤特异性突变的下一代测序（NGS）的准确性受到PCR/测序期间引入的错误、由DNA损伤引起的碱基取代以及正常细胞中以低频率存在的预先存在的突变的阻碍。在这里，我们使用分子条形码对血浆DNA（平均4.5ng）中与胰腺癌相关的基因（包含2.8kb的基因组DNA）进行了NGS。平均测序分子数为900个，每个分子的测序深度为100个或更多。我们还开发了一种生物信息学变体过滤器，称为CV 78，以去除不被认为是肿瘤特异性的变体，即，那些在癌症体细胞突变目录数据库中不存在或以低频率出现的突变。在一个包括57名胰腺癌患者和12名健康个体的队列中，测序最初分别在31名（54%）和5名（42%）中鉴定出变异，而在应用CV 78过滤器后，19名（33%）和0名为变异阳性。在由86名胰腺癌患者和20名导管内乳头状粘液性肿瘤（IPMN）患者组成的验证队列中，62名（72%）胰腺癌患者和10名（50%）IPMN患者最初为变异阳性。CV 78过滤后，这些值分别减少至32（37%）和1（5%）。血浆中过滤变体的变体等位基因频率范围为0.25%至76.1%。因此，将NGS和分子条形码与随后的过滤相结合可能会消除大多数非肿瘤特异性突变。
The accuracy of next-generation sequencing (NGS) for detecting tumor-specific mutations in plasma DNA is hindered by errors introduced during PCR/sequencing, base substitutions caused by DNA damage, and pre-existing mutations in normal cells that are present at a low frequency. Here, we performed NGS of genes related to pancreatic cancer (comprising 2.8 kb of genomic DNA) in plasma DNA (average 4.5 ng) using molecular barcodes. The average number of sequenced molecules was 900, and the sequencing depth per molecule was 100 or more. We also developed a bioinformatic variant filter, called CV78, to remove variants that were not considered to be tumor-specific, i.e., those that are either absent or occur at low frequencies in the Catalogue of Somatic Mutations in Cancer database. In a cohort comprising 57 pancreatic cancer patients and 12 healthy individuals, sequencing initially identified variants in 31 (54%) and 5 (42%), respectively, whereas after applying the CV78 filter, 19 (33%) and zero were variant-positive. In a validation cohort consisting of 86 patients with pancreatic cancer and 20 patients with intraductal papillary mucinous neoplasm (IPMN), 62 (72%) with pancreatic cancer patients and 10 (50%) IPMN patients were initially variant positive. After CV78 filtering, these values were reduced to 32 (37%) and 1 (5%), respectively. The variant allele frequency of filtered variants in plasma ranged from 0.25% to 76.1%. Therefore, combining NGS and molecular barcodes with subsequent filtering is likely to eliminate most non-tumor-specific mutations.
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影响因子：
64.8
作者：
通讯作者：
--
DOI：
10.1073/pnas.1208715109
发表时间：
2012-09-04
影响因子：
11.1
作者：
Schmitt, Michael W.;Kennedy, Scott R.;Loeb, Lawrence A.
通讯作者：
Loeb, Lawrence A.
影响因子：
46.9
作者：
Newman AM;Lovejoy AF;Klass DM;Kurtz DM;Chabon JJ;Scherer F;Stehr H;Liu CL;Bratman SV;Say C;Zhou L;Carter JN;West RB;Sledge GW;Shrager JB;Loo BW Jr;Neal JW;Wakelee HA;Diehn M;Alizadeh AA
通讯作者：
Alizadeh AA
影响因子：
14.9
作者：
Forbes SA;Bindal N;Bamford S;Cole C;Kok CY;Beare D;Jia M;Shepherd R;Leung K;Menzies A;Teague JW;Campbell PJ;Stratton MR;Futreal PA
通讯作者：
Futreal PA
影响因子：
4.4
作者：
Xu C;Nezami Ranjbar MR;Wu Z;DiCarlo J;Wang Y
通讯作者：
Wang Y