Dataset Information

Somatic Mutation Screening Using Archival Formalin-Fixed, Paraffin-Embedded Tissues by Fluidigm Multiplex PCR and Illumina Sequencing.

ABSTRACT: 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.

SUBMITTER: Wang M

PROVIDER: S-EPMC4597275 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Publications

Somatic Mutation Screening Using Archival Formalin-Fixed, Paraffin-Embedded Tissues by Fluidigm Multiplex PCR and Illumina Sequencing.

Wang Ming M Escudero-Ibarz Leire L Moody Sarah S Zeng Naiyan N Clipson Alexandra A Huang Yuanxue Y Xue Xuemin X Grigoropoulos Nicholas F NF Barrans Sharon S Worrillow Lisa L Forshew Tim T Su Jing J Firth Andrew A Martin Howard H Jack Andrew A Brugger Kim K Du Ming-Qing MQ

The Journal of molecular diagnostics : JMD 20150709 5

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 ...[more]

PMID: 26165823

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Project description:There is increasing acceptance of the critical importance of correlating the morphologic features of tissue with the data obtained from various molecular analytic techniques. Access to archived formalin-fixed and paraffin-embedded (FFPE) tissue specimens via shotgun-based proteomic analyses may, therefore, open new avenues for both prospective and retrospective translational research. However, one of the remaining issues in performing comparative proteomic measurements among FFPE tissues relates to potential variability in protein composition and retrieval based on length of storage periods. Optimized protein extraction and digestion procedures for handling FFPE tissues are coupled with the capillary isotachophoresis-based proteome technology to evaluate the effects of length of storage period on archival tissue proteome analysis across 10 archived uterine mesenchymal tumor tissue blocks, including 9 uterine leiomyomas dating from 1990 to 2002 and a single case of alveolar soft part sarcoma (ASPS) from 1980. Several statistical measures, including the Pearson correlation coefficient, coefficient of variance, k-means clustering, and ANOVA, are employed to evaluate the possibility of an archival effect on individual proteins or groups of proteins within nine leiomyomas. Low abundance proteins may be more susceptible to the long-term storage as these proteins are more difficult to be retrieved and extracted as the tissue block ages in paraffin. Despite using tissue blocks stored for as many as 28 years, high confidence and comparative proteome analysis between the leiomyomas and the sarcoma is achieved. Though sharing over 1800 common proteins in a core set, a total of 80 proteins unique to the sarcoma are identified distinguishing the ASPS from the leiomyomas. Vacuolar proton translocating ATPase 116 kDa subunit isoform a3, one of the unique proteins expressed in the ASPS, is further validated by immunohistochemistry (IHC). Although IHC is highly sensitive and provides the subcellular resolution, mass spectrometry-based proteome profiling enables global identification and quantification of thousands of proteins without a priori knowledge of individual proteins being analyzed or the need of validated antibodies.

Dataset Information

Somatic Mutation Screening Using Archival Formalin-Fixed, Paraffin-Embedded Tissues by Fluidigm Multiplex PCR and Illumina Sequencing.

Publications

Somatic Mutation Screening Using Archival Formalin-Fixed, Paraffin-Embedded Tissues by Fluidigm Multiplex PCR and Illumina Sequencing.

Similar Datasets

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