scholarly journals MicroSEC: Sequence error filtering pipeline for formalin-fixed and paraffin-embedded samples

2021 ◽  
Author(s):  
Masachika Ikegami ◽  
Shinji Kohsaka ◽  
Takeshi Hirose ◽  
Toshihide Ueno ◽  
Naoki Kanomata ◽  
...  
Keyword(s):  
Uneven Distribution ◽  
Clinical Sequencing ◽  
Sequencing Errors ◽  
Sequence Error ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Fresh Frozen ◽  
Formalin Fixed ◽  
Error Filtering

Abstract The clinical sequencing of tumors is usually performed on formalin-fixed, paraffin-embedded (FFPE) samples and results in many sequencing errors. Most of these errors are detected in chimeric reads caused by single-strand DNA molecules with microhomology. Our filtering pipeline, MicroSEC, focuses on the uneven distribution of mutations in each read and removes the sequencing errors in FFPE samples without eliminating the true mutations that are also detected in fresh frozen samples.

scholarly journals MicroSEC filters sequence errors for formalin-fixed and paraffin-embedded samples

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Masachika Ikegami ◽  
Shinji Kohsaka ◽  
Takeshi Hirose ◽  
Toshihide Ueno ◽  
Satoshi Inoue ◽  
...  
Keyword(s):  
Confidence Interval ◽  
Single Strand ◽  
Dna Molecules ◽  
Clinical Sequencing ◽  
Sequencing Errors ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Fresh Frozen ◽  
The Individual ◽  
Formalin Fixed

AbstractThe clinical sequencing of tumors is usually performed on formalin-fixed, paraffin-embedded samples and results in many sequencing errors. We identified that most of these errors are detected in chimeric reads caused by single-strand DNA molecules with microhomology. During the end-repair step of library preparation, mutations are introduced by the mis-annealing of two single-strand DNA molecules comprising homologous sequences. The mutated bases are distributed unevenly near the ends in the individual reads. Our filtering pipeline, MicroSEC, focuses on the uneven distribution of mutations in each read and removes the sequencing errors in formalin-fixed, paraffin-embedded samples without over-eliminating the mutations detected also in fresh frozen samples. Amplicon-based sequencing using 97 mutations confirmed that the sensitivity and specificity of MicroSEC were 97% (95% confidence interval: 82–100%) and 96% (95% confidence interval: 88–99%), respectively. Our pipeline will increase the reliability of the clinical sequencing and advance the cancer research using formalin-fixed, paraffin-embedded samples.

scholarly journals Exome Sequencing of Fresh-frozen or Formalin-fixed Paraffin-embedded B6C3F1/N Mouse Hepatocellular Carcinomas Arising Either Spontaneously or due to Chronic Chemical Exposure

2018 ◽  
Vol 46 (6) ◽  
pp. 706-718 ◽  
Author(s):  
Scott S. Auerbach ◽  
Miaofei Xu ◽  
B. Alex Merrick ◽  
Mark J. Hoenerhoff ◽  
Dhiral Phadke ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Chemical Exposure ◽  
Mutational Spectra ◽  
Gingko Biloba ◽  
Novel Approach ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Fresh Frozen ◽  
Formalin Fixed

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide; however, the mutational properties of HCC-associated carcinogens remain largely uncharacterized. We hypothesized that mechanisms underlying chemical-induced HCC can be characterized by evaluating the mutational spectra of these tumors. To test this hypothesis, we performed exome sequencing of B6C3F1/N HCCs that arose either spontaneously in vehicle controls ( n = 3) or due to chronic exposure to gingko biloba extract (GBE; n = 4) or methyleugenol (MEG; n = 3). Most archived tumor samples are available as formalin-fixed paraffin-embedded (FFPE) blocks, rather than fresh-frozen (FF) samples; hence, exome sequencing from paired FF and FFPE samples was compared. FF and FFPE samples showed 63% to 70% mutation concordance. Multiple known (e.g., Ctnnb1T41A, BrafV637E) and novel (e.g., Erbb4C559S, Card10A700V, and Klf11P358L) mutations in cancer-related genes were identified. The overall mutational burden was greater for MEG than for GBE or spontaneous HCC samples. To characterize the mutagenic mechanisms, we analyzed the mutational spectra in the HCCs according to their trinucleotide motifs. The MEG tumors clustered closest to Catalogue of Somatic Mutations in Cancer signatures 4 and 24, which are, respectively, associated with benzo(a)pyrene- and aflatoxin-induced HCCs in humans. These results establish a novel approach for classifying liver carcinogens and understanding the mechanisms of hepatocellular carcinogenesis.

scholarly journals 3′RNA Sequencing Accurately Classifies Formalin-Fixed Paraffin-Embedded Uterine Leiomyomas

Cancers ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3839
Author(s):  
Miika Mehine ◽  
Sara Khamaiseh ◽  
Terhi Ahvenainen ◽  
Tuomas Heikkinen ◽  
Anna Äyräväinen ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Cost Effective ◽  
Uterine Leiomyomas ◽  
Reproductive Age ◽  
Genetic Changes ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Fresh Frozen ◽  
Formalin Fixed

Uterine leiomyomas are benign smooth muscle tumors occurring in 70% of women of reproductive age. The majority of leiomyomas harbor one of three well-established genetic changes: a hotspot mutation in MED12, overexpression of HMGA2, or biallelic loss of FH. The majority of studies have classified leiomyomas by complex and costly methods, such as whole-genome sequencing, or by combining multiple traditional methods, such as immunohistochemistry and Sanger sequencing. The type of specimens and the amount of resources available often determine the choice. A more universal, cost-effective, and scalable method for classifying leiomyomas is needed. The aim of this study was to evaluate whether RNA sequencing can accurately classify formalin-fixed paraffin-embedded (FFPE) leiomyomas. We performed 3′RNA sequencing with 44 leiomyoma and 5 myometrium FFPE samples, revealing that the samples clustered according to the mutation status of MED12, HMGA2, and FH. Furthermore, we confirmed each subtype in a publicly available fresh frozen dataset. These results indicate that a targeted 3′RNA sequencing panel could serve as a cost-effective and robust tool for stratifying both fresh frozen and FFPE leiomyomas. This study also highlights 3′RNA sequencing as a promising method for studying the abundance of unexploited tissue material that is routinely stored in hospital archives.

scholarly journals Multiclass cancer classification in fresh frozen and formalin-fixed paraffin-embedded tissue by DigiWest multiplex protein analysis

2020 ◽  
Vol 100 (10) ◽  
pp. 1288-1299
Author(s):  
Teresa Bockmayr ◽  
Gerrit Erdmann ◽  
Denise Treue ◽  
Philipp Jurmeister ◽  
Julia Schneider ◽  
...  
Keyword(s):  
Squamous Cell ◽  
Squamous Cell Carcinomas ◽  
Cancer Classification ◽  
Protein Profiling ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Fresh Frozen ◽  
Formalin Fixed

Abstract Histomorphology and immunohistochemistry are the most common ways of cancer classification in routine cancer diagnostics, but often reach their limits in determining the organ origin in metastasis. These cancers of unknown primary, which are mostly adenocarcinomas or squamous cell carcinomas, therefore require more sophisticated methodologies of classification. Here, we report a multiplex protein profiling-based approach for the classification of fresh frozen and formalin-fixed paraffin-embedded (FFPE) cancer tissue samples using the digital western blot technique DigiWest. A DigiWest-compatible FFPE extraction protocol was developed, and a total of 634 antibodies were tested in an initial set of 16 FFPE samples covering tumors from different origins. Of the 303 detected antibodies, 102 yielded significant correlation of signals in 25 pairs of fresh frozen and FFPE primary tumor samples, including head and neck squamous cell carcinomas (HNSC), lung squamous cell carcinomas (LUSC), lung adenocarcinomas (LUAD), colorectal adenocarcinomas (COAD), and pancreatic adenocarcinomas (PAAD). For this signature of 102 analytes (covering 88 total proteins and 14 phosphoproteins), a support vector machine (SVM) algorithm was developed. This allowed for the classification of the tissue of origin for all five tumor types studied here with high overall accuracies in both fresh frozen (90.4%) and FFPE (77.6%) samples. In addition, the SVM classifier reached an overall accuracy of 88% in an independent validation cohort of 25 FFPE tumor samples. Our results indicate that DigiWest-based protein profiling represents a valuable method for cancer classification, yielding conclusive and decisive data not only from fresh frozen specimens but also FFPE samples, thus making this approach attractive for routine clinical applications.

scholarly journals Confirming putative variants at ≤ 5% allele frequency using allele enrichment and Sanger sequencing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yan Helen Yan ◽  
Sherry X. Chen ◽  
Lauren Y. Cheng ◽  
Alyssa Y. Rodriguez ◽  
Rui Tang ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Limit Of Detection ◽  
High Sensitivity ◽  
Sequencing Errors ◽  
Whole Exome ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Fresh Frozen ◽  
The Cost ◽  
Allelic Fraction

AbstractWhole exome sequencing (WES) is used to identify mutations in a patient’s tumor DNA that are predictive of tumor behavior, including the likelihood of response or resistance to cancer therapy. WES has a mutation limit of detection (LoD) at variant allele frequencies (VAF) of 5%. Putative mutations called at ≤ 5% VAF are frequently due to sequencing errors, therefore reporting these subclonal mutations incurs risk of significant false positives. Here we performed ~ 1000 × WES on fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissue biopsy samples from a non-small cell lung cancer patient, and identified 226 putative mutations at between 0.5 and 5% VAF. Each variant was then tested using NuProbe NGSure, to confirm the original WES calls. NGSure utilizes Blocker Displacement Amplification to first enrich the allelic fraction of the mutation and then uses Sanger sequencing to determine mutation identity. Results showed that 52% of the 226 (117) putative variants were disconfirmed, among which 2% (5) putative variants were found to be misidentified in WES. In the 66 cancer-related variants, the disconfirmed rate was 82% (54/66). This data demonstrates Blocker Displacement Amplification allelic enrichment coupled with Sanger sequencing can be used to confirm putative mutations ≤ 5% VAF. By implementing this method, next-generation sequencing can reliably report low-level variants at a high sensitivity, without the cost of high sequencing depth.

scholarly journals The impact of RNA extraction method on accurate RNA sequencing from formalin-fixed paraffin-embedded tissues

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Michal Marczyk ◽  
Chunxiao Fu ◽  
Rosanna Lau ◽  
Lili Du ◽  
Alexander J. Trevarton ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Rna Sequencing ◽  
Rna Extraction ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
The Impact ◽  
Formalin Fixed

Abstract Background Utilization of RNA sequencing methods to measure gene expression from archival formalin-fixed paraffin-embedded (FFPE) tumor samples in translational research and clinical trials requires reliable interpretation of the impact of pre-analytical variables on the data obtained, particularly the methods used to preserve samples and to purify RNA. Methods Matched tissue samples from 12 breast cancers were fresh frozen (FF) and preserved in RNAlater or fixed in formalin and processed as FFPE tissue. Total RNA was extracted and purified from FF samples using the Qiagen RNeasy kit, and in duplicate from FFPE tissue sections using three different kits (Norgen, Qiagen and Roche). All RNA samples underwent whole transcriptome RNA sequencing (wtRNAseq) and targeted RNA sequencing for 31 transcripts included in a signature of sensitivity to endocrine therapy. We assessed the effect of RNA extraction kit on the reliability of gene expression levels using linear mixed-effects model analysis, concordance correlation coefficient (CCC) and differential analysis. All protein-coding genes in the wtRNAseq and three gene expression signatures for breast cancer were assessed for concordance. Results Despite variable quality of the RNA extracted from FFPE samples by different kits, all had similar concordance of overall gene expression from wtRNAseq between matched FF and FFPE samples (median CCC 0.63–0.66) and between technical replicates (median expression difference 0.13–0.22). More than half of genes were differentially expressed between FF and FFPE, but with low fold change (median |LFC| 0.31–0.34). Two out of three breast cancer signatures studied were highly robust in all samples using any kit, whereas the third signature was similarly discordant irrespective of the kit used. The targeted RNAseq assay was concordant between FFPE and FF samples using any of the kits (CCC 0.91–0.96). Conclusions The selection of kit to purify RNA from FFPE did not influence the overall quality of results from wtRNAseq, thus variable reproducibility of gene signatures probably relates to the reliability of individual gene selected and possibly to the algorithm. Targeted RNAseq showed promising performance for clinical deployment of quantitative assays in breast cancer from FFPE samples, although numerical scores were not identical to those from wtRNAseq and would require calibration.

scholarly journals Time and tumor type (primary or metastatic) do not influence the detection of BRAF/NRAS mutations in formalin fixed paraffin embedded samples from melanomas

2016 ◽  
Vol 54 (11) ◽  
Author(s):  
Miriam Potrony ◽  
Celia Badenas ◽  
Bénédicte Naerhuyzen ◽  
Paula Aguilera ◽  
Joan Anton Puig-Butille ◽  
...  
Keyword(s):  
Melanoma Patient ◽  
Primary Melanoma ◽  
Sentinel Lymph Nodes ◽  
Tumor Type ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Complete Sequencing ◽  
Formalin Fixed ◽  
Tumor Area

AbstractBackground:Methods:DNA was obtained from 144 FFPE samples (62 primary melanoma, 43 sentinel lymph nodes [SLN] and 39 metastasis).Results:Complete sequencing results were obtained from 75% (108/144) of the samples, and at least one gene was sequenced in 89% (128/144) of them.Conclusions:Preserving sufficient tumor area in FFPE blocks is important. It is necessary to keep the FFPE blocks, no matter their age, as they are necessary to decide the best treatment for the melanoma patient.

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