Somatic Mutation Screening Using Archival Formalin-Fixed, Paraffin-Embedded Tissues by Fluidigm Multiplex PCR and Illumina Sequencing
| Autor: | Yuanxue Huang, Tim Forshew, Jing Su, Sarah Moody, Alexandra Clipson, Leire Escudero-Ibarz, Nicholas Francis Grigoropoulos, Xuemin Xue, Kim Brügger, Ming-Qing Du, Andrew Jack, Naiyan Zeng, Lisa Worrillow, Ming Wang, Sharon Barrans, Andrew E. Firth, Howard Martin |
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| Přispěvatelé: | Firth, Andrew [0000-0002-7986-9520], Apollo - University of Cambridge Repository |
| Rok vydání: | 2015 |
| Předmět: |
Tissue Fixation
Lymphoma DNA Mutational Analysis Biology Pathology and Forensic Medicine symbols.namesake chemistry.chemical_compound Germline mutation INDEL Mutation Formaldehyde Multiplex polymerase chain reaction Humans False Positive Reactions Genetic Testing Indel Allele frequency Gene Illumina dye sequencing Sanger sequencing Genetics Paraffin Embedding High-Throughput Nucleotide Sequencing Regular Article chemistry Amino Acid Substitution symbols Molecular Medicine Multiplex Polymerase Chain Reaction Taq polymerase Algorithms |
| Zdroj: | The Journal of molecular diagnostics : JMD. 17(5) |
| ISSN: | 1943-7811 |
| Popis: | High-throughput somatic mutation screening using FFPE tissues is a major challenge because of a lack of established methods and validated variant calling algorithms. We aimed to develop a targeted sequencing protocol by Fluidigm multiplex PCR and Illumina sequencing and to establish a companion variant calling algorithm. The experimental protocol and variant calling algorithm were first developed and optimized against a series of somatic mutations (147 substitutions, 12 indels ranging from 1 to 33 bp) in seven genes, previously detected by Sanger sequencing of DNA from 163 FFPE lymphoma biopsy specimens. The optimized experimental protocol and variant calling algorithm were further ascertained in two separate experiments by including the seven genes as a part of larger gene panels (22 or 13 genes) using FFPE and high-molecular-weight lymphoma DNAs, respectively. We found that most false-positive variants were due to DNA degradation, deamination, and Taq polymerase errors, but they were nonreproducible and could be efficiently eliminated by duplicate experiments. A small fraction of false-positive variants appeared in duplicate, but they were at low alternative allele frequencies and could be separated from mutations when appropriate threshold value was used. In conclusion, we established a robust practical approach for high-throughput mutation screening using archival FFPE tissues. |
| Databáze: | OpenAIRE |
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