Single-locus enrichment without amplification for sequencing and direct detection of epigenetic modifications

详细信息    查看全文

• 作者：Thang T. Pham ; Jun Yin ; John S. Eid ; Evan Adams…
• 刊名：Molecular Genetics and Genomics
• 出版年：2016
• 出版时间：June 2016
• 年：2016
• 卷：291
• 期：3
• 页码：1491-1504
• 全文大小：1,616 KB
• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Life Sciences
  Cell Biology
  Biochemistry
  Microbial Genetics and Genomics
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1617-4623
• 卷排序：291

文摘

A gene-level targeted enrichment method for direct detection of epigenetic modifications is described. The approach is demonstrated on the CGG-repeat region of the FMR1 gene, for which large repeat expansions, hitherto refractory to sequencing, are known to cause fragile X syndrome. In addition to achieving a single-locus enrichment of nearly 700,000-fold, the elimination of all amplification steps removes PCR-induced bias in the repeat count and preserves the native epigenetic modifications of the DNA. In conjunction with the single-molecule real-time sequencing approach, this enrichment method enables direct readout of the methylation status and the CGG repeat number of the FMR1 allele(s) for a clonally derived cell line. The current method avoids potential biases introduced through chemical modification and/or amplification methods for indirect detection of CpG methylation events.KeywordsTargeted enrichmentSingle molecule sequencingFMR1Fragile X syndromeEpigenetic modificationTandem repeats