scholarly journals Bioinformatics and DNA-extraction strategies to reliably detect genetic variants from FFPE breast tissue samples

2019 ◽  
Author(s):  
Aditya Vijay Bhagwate ◽  
Yuanhang Liu ◽  
Stacey J. Winham ◽  
Samantha J. McDonough ◽  
Melody L. Stallings-Mann ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Genetic Variants ◽  
Variant Calling ◽  
Allelic Frequency ◽  
Tissue Samples ◽  
Mutational Signature ◽  
Ffpe Samples ◽  
Fresh Frozen ◽  
Wet Lab ◽  
Control Samples

Abstract Background Archived formalin fixed paraffin embedded (FFPE) samples are valuable clinical resources to examine clinically relevant morphology features and also to study genetic changes. However, DNA quality and quantity of FFPE samples are often sub-optimal, and resulting NGS-based genetics variant detections are prone to false positives. Evaluations of wet-lab and bioinformatics approaches are needed to optimize variant detection from FFPE samples. Results As a pilot study, we designed within-subject triplicate samples of DNA derived from paired FFPE and fresh frozen breast tissues to highlight FFPE-specific artifacts. For FFPE samples, we tested two FFPE DNA extraction methods to determine impact of wet-lab procedures on variant calling: QIAGEN QIAamp DNA Mini Kit ("QA"), and QIAGEN GeneRead DNA FFPE Kit ("QGR"). We also used negative-control (NA12891) and positive control samples (Horizon Discovery Reference Standard FFPE). All DNA sample libraries were prepared for NGS according to the QIAseq Human Breast Cancer Targeted DNA Panel protocol and sequenced on the HiSeq 4000. Variant calling and filtering were performed using QIAGEN Gene Globe Data Portal. Detailed variant concordance comparisons and mutational signature analysis were performed to investigate effects of FFPE samples compared to paired fresh frozen samples, along with different library preparations. In this study, we found that five times or more variants were called with FFPE samples, compared to their paired fresh-frozen tissue samples even after applying molecular barcoding error-correction and default bioinformatics filtering recommended by the vendor. We also found that QGR as an optimized FFPE-DNA extraction approach leads to much fewer discordant variants between paired fresh frozen and FFPE samples. Approximately 92% of the uniquely called FFPE variants were of low allelic frequency range (<5%), and collectively shared a “C>T|G>A” mutational signature known to be representative of FFPE artifacts resulting fromcytosine deamination. Based on control samples and FFPE-frozen replicates, we derived an effective filtering strategy with associated empirical false-discovery estimates. Conclusions Through this study, we demonstrated feasibility of calling and filtering genetic variants from FFPE tissue samples using a combined strategy with molecular barcodes, optimized DNA extraction, and bioinformatics methods incorporating genomics context such as mutational signature and variant allelic frequency.

scholarly journals Bioinformatics and DNA-extraction strategies to reliably detect genetic variants from FFPE breast tissue samples

2019 ◽  
Author(s):  
Aditya Vijay Bhagwate ◽  
Yuanhang Liu ◽  
Stacey J. Winham ◽  
Samantha J. McDonough ◽  
Melody L. Stallings-Mann ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Genetic Variants ◽  
Variant Calling ◽  
Allelic Frequency ◽  
Tissue Samples ◽  
Mutational Signature ◽  
Ffpe Samples ◽  
Fresh Frozen ◽  
Wet Lab ◽  
Control Samples

Abstract Background Archived formalin fixed paraffin embedded (FFPE) samples are valuable clinical resources to examine clinically relevant morphology features and also to study genetic changes. However, DNA quality and quantity of FFPE samples are often sub-optimal, and resulting NGS-based genetics variant detections are prone to false positives. Evaluations of wet-lab and bioinformatics approaches are needed to optimize variant detection from FFPE samples. Results As a pilot study, we designed within-subject triplicate samples of DNA derived from paired FFPE and fresh frozen breast tissues to highlight FFPE-specific artifacts. For FFPE samples, we tested two FFPE DNA extraction methods to determine impact of wet-lab procedures on variant calling: QIAGEN QIAamp DNA Mini Kit ("QA"), and QIAGEN GeneRead DNA FFPE Kit ("QGR"). We also used negative-control (NA12891) and positive control samples (Horizon Discovery Reference Standard FFPE). All DNA sample libraries were prepared for NGS according to the QIAseq Human Breast Cancer Targeted DNA Panel protocol and sequenced on the HiSeq 4000. Variant calling and filtering were performed using QIAGEN Gene Globe Data Portal. Detailed variant concordance comparisons and mutational signature analysis were performed to investigate effects of FFPE samples compared to paired fresh frozen samples, along with different library preparations. In this study, we found that five times or more variants were called with FFPE samples, compared to their paired fresh-frozen tissue samples even after applying molecular barcoding error-correction and default bioinformatics filtering recommended by the vendor. We also found that QGR as an optimized FFPE-DNA extraction approach leads to much fewer discordant variants between paired fresh frozen and FFPE samples. Approximately 92% of the uniquely called FFPE variants were of low allelic frequency range (<5%), and collectively shared a “C>T|G>A” mutational signature known to be representative of FFPE artifacts resulting fromcytosine deamination. Based on control samples and FFPE-frozen replicates, we derived an effective filtering strategy with associated empirical false-discovery estimates. Conclusions Through this study, we demonstrated feasibility of calling and filtering genetic variants from FFPE tissue samples using a combined strategy with molecular barcodes, optimized DNA extraction, and bioinformatics methods incorporating genomics context such as mutational signature and variant allelic frequency.

scholarly journals Bioinformatics and DNA-extraction strategies to reliably detect genetic variants from FFPE breast tissue samples

2019 ◽  
Author(s):  
Aditya Vijay Bhagwate ◽  
Yuanhang Liu ◽  
Stacey J. Winham ◽  
Samantha J. McDonough ◽  
Melody L. Stallings-Mann ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Variant Calling ◽  
Extraction Methods ◽  
Allelic Frequency ◽  
Tissue Samples ◽  
Mutational Signature ◽  
Ffpe Samples ◽  
Fresh Frozen ◽  
Wet Lab ◽  
Dna Extraction Methods

Abstract Background: Archived formalin fixed paraffin embedded (FFPE) samples are valuable clinical resources to examine clinically relevant morphology features and also to study genetic changes. However, DNA quality and quantity of FFPE samples are sub-optimal, and resulting NGS-based genetics variant detections are prone to false positives. Evaluations of wet-lab and bioinformatics approaches are needed to optimize variant detection from FFPE samples. Results: As a pilot study, we designed within-subject triplicate samples of DNA derived from paired FFPE and fresh frozen breast tissues to highlight FFPE-specific artifacts. For FFPE samples, we tested two FFPE DNA extraction methods to determine impact of wet-lab procedures on variant calling: QIAGEN QIAamp DNA Mini Kit ("QA"), and QIAGEN GeneRead DNA FFPE Kit ("QGR"). We also used negative-control (NA12891) and positive control samples (Horizon Discovery Reference Standard FFPE). All DNA sample libraries were prepared for NGS according to the QIAseq Human Breast Cancer Targeted DNA Panel protocol and sequenced on the HiSeq 4000. Variant calling and filtering were performed using QIAGEN Gene Globe Data Portal. Detailed variant concordance comparisons and mutational signature analysis were performed to investigate effects of FFPE samples compared to paired fresh frozen samples, along with different DNA extraction methods. In this study, we found that five times or more variants were called with FFPE samples, compared to their paired fresh-frozen tissue samples even after applying molecular barcoding error-correction and default bioinformatics filtering recommended by the vendor. We also found that QGR as an optimized FFPE-DNA extraction approach leads to much fewer discordant variants between paired fresh frozen and FFPE samples. Approximately 92 % of the uniquely called FFPE variants were of low allelic frequency range (<5%), and collectively shared a “C>T|G>A” mutational signature known to be representative of FFPE artifacts resulting from cytosine deamination. Based on control samples and FFPE-frozen replicates, we derived an effective filtering strategy with associated empirical false-discovery estimates. Conclusions: Through this study, we demonstrated feasibility of calling and filtering genetic variants from FFPE tissue samples using a combined strategy with molecular barcodes, optimized DNA extraction, and bioinformatics methods incorporating genomics context such as mutational signature and variant allelic frequency.

scholarly journals Nested PCR for the Diagnosis of Feline Sporotrichosis From Formalin-Fixed and Paraffin-Embedded Samples Using Different DNA Extraction Protocols

2022 ◽  
Vol 8 ◽  
Author(s):  
Raul Leal Faria Luiz ◽  
Rodrigo Caldas Menezes ◽  
Sandro Antonio Pereira ◽  
Raquel de Vasconcellos Carvalhaes de Oliveira ◽  
Manoel Marques Evangelista Oliveira
Keyword(s):  
Dna Extraction ◽  
Nested Pcr ◽  
Paraffin Section ◽  
Chemical Extraction ◽  
Dimorphic Fungi ◽  
Tissue Samples ◽  
Ffpe Samples ◽  
Formalin Fixed ◽  
Skin Samples ◽  
Positive Controls

Sporotrichosis is a chronic, cosmopolitan granulomatous mycosis that affects humans and animals. The infection is caused by the dimorphic fungi Sporothrix sp. The aims of the present study were to evaluate, standardize and validate a nested PCR technique using two DNA purification kits for the extraction of DNA from formalin fixed and paraffin-embedded tissues (FFPE) for Sporothrix sp. detection. FFPE mycological culture pellet samples of different Sporothrix species (S. chilensis, S. mexicana, S. pallida, S. globosa, S. brasiliensis and S. schenckii) were used as positive controls and clinical FFPE tissue samples of animals positive for Cryptococcus sp., Leishmania infantum and Histoplasma sp. were used as negative controls. Ten clinical FFPE skin samples from cats with sporotrichosis were used to validate the nested PCR. These samples were cut into two distinct paraffin sectioning protocols (5 and 16 μm thick). The paraffin sections were subjected to two different DNA extraction kits (chemical and thermal extractions). A nested PCR was performed on the extracted DNA to identify the genus Sporothrix. The chemical extraction protocol with the 5 μm thick paraffin section was more effective in extracting DNA from Sporothrix sp. from FFPE samples and the nested PCR technique showed the highest sensitivities (100% in the positive controls and of 50% in the skin samples of cats) and specificity (100%). Therefore, the nested PCR using this protocol has great potential to be applied in Sporothrix sp. diagnosis in FFPE samples of cats.

Enabling variant calling in challenging FFPE samples by coupling a novel library preparation chemistry with exome sequencing.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e15584-e15584
Author(s):  
Bosun Min ◽  
Ushati Das Chakravarty ◽  
Manqing Hong ◽  
Ekaterina Star ◽  
Hsiao-Yun Huang ◽  
...  
Keyword(s):  
Target Genes ◽  
Variant Calling ◽  
Library Preparation ◽  
Tissue Samples ◽  
Dna Library ◽  
Oncology Research ◽  
Dna Libraries ◽  
Wide Range ◽  
Ffpe Samples ◽  
High Yields

e15584 Background: Comprehensive tumor profiling using NGS is fundamentally transforming oncology research. However, converting archival tissue samples into libraries is often challenging due to the low quantity and quality of DNA. Here we present accurate detection of variants in the human exome using the novel chemistry of the xGen Prism DNA library preparation kit, optimized for low-input and degraded samples, with xGen Research Exome v2.0 hybrid-capture enrichment. Methods: The IDT Exome v2 panel was used to carry out targeted sequencing of Prism DNA libraries generated from archival FFPE samples. The unique library preparation is enabled by an engineered mutant ligase and proprietary adapters that prevent chimeras and suppress dimer-formation, thereby maximizing the conversion of input DNA to sequencing libraries. Results: We achieved high yields of library (300-400 ng) from input amounts as low as 25 ng for severely damaged FFPE samples (DIN 1-3), > 90% on-target rates and uniform depth of coverage ( > 96% bases covered at > 20X and > 98% bases covered at > 10X) for FFPE samples across a wide range in quality. We also observed minimal exon drop-outs in difficult-to-target genes for severely damaged FFPE material. To validate the variant calling performance of the Prism-Exome workflow, we used the Horizon OncoSpan FFPE reference control which contains 1-92% AF SNVs and Indels and achieved > 98% sensitivity across ~250 SNVs and Indels. Conclusions: This study demonstrates that the xGen Exome Research v2, when combined with xGen Prism DNA library preparation, provides researchers with a complete human exome FFPE-sequencing solution with robust performance across FFPE samples of varying quality.

scholarly journals Bioinformatics and DNA-extraction strategies to reliably detect genetic variants from FFPE breast tissue samples

BMC Genomics ◽  
2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Aditya Vijay Bhagwate ◽  
Yuanhang Liu ◽  
Stacey J. Winham ◽  
Samantha J. McDonough ◽  
Melody L. Stallings-Mann ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Genetic Variants ◽  
Breast Tissue ◽  
Tissue Samples

scholarly journals High-throughput proteomic analysis of FFPE tissue samples facilitates tumor stratification

2019 ◽  
Author(s):  
Yi Zhu ◽  
Tobias Weiss ◽  
Qiushi Zhang ◽  
Rui Sun ◽  
Bo Wang ◽  
...  
Keyword(s):  
Biomarker Discovery ◽  
Temporal Stability ◽  
B Cell Lymphoma ◽  
Ffpe Tissue ◽  
Tissue Samples ◽  
Pattern Similarity ◽  
Ffpe Samples ◽  
Fresh Frozen ◽  
Peroxidase Enzyme ◽  
High Degree

AbstractFormalin-fixed, paraffin-embedded (FFPE), biobanked tissue samples offer an invaluable resource for clinical and biomarker research. Here we developed a pressure cycling technology (PCT)-SWATH mass spectrometry workflow to analyze FFPE tissue proteomes and applied it to the stratification of prostate cancer (PCa) and diffuse large B-cell lymphoma (DLBCL) samples. We show that the proteome patterns of FFPE PCa tissue samples and their analogous fresh frozen (FF) counterparts have a high degree of similarity and we confirmed multiple proteins consistently regulated in PCa tissues in an independent sample cohort. We further demonstrate temporal stability of proteome patterns from FFPE samples that were stored between one to 15 years in a biobank and show a high degree of the proteome pattern similarity between two types histological region of small FFPE samples, i.e. punched tissue biopsies and thin tissue sections of micrometer thickness, despite the existence of certain degree of biological variations. Applying the method to two independent DLBCL cohorts we identified myeloperoxidase (MPO), a peroxidase enzyme, as a novel prognostic marker. In summary, this study presents a robust proteomic method to analyze bulk and biopsy FFPE tissues and reports the first systematic comparison of proteome maps generated from FFPE and FF samples. Our data demonstrate the practicality and superiority of FFPE over FF samples for proteome in biomarker discovery. Promising biomarker candidates for PCa and DLBCL have been discovered.

scholarly journals Comparative analysis of somatic variant calling on matched FF and FFPE WGS from a metastatic prostate sample

2019 ◽  
Author(s):  
Louise de Schaetzen van Brienen ◽  
Maarten Larmuseau ◽  
Kim Van der Eecken ◽  
Jan Fostier ◽  
Piet Ost ◽  
...  
Keyword(s):  
Cohort Studies ◽  
Large Fraction ◽  
Variant Calling ◽  
Metastatic Tumor ◽  
Ffpe Sample ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Ffpe Samples ◽  
Fresh Frozen ◽  
Ffpe Material

Abstract Background. Research grade Fresh Frozen (FF) DNA material is not yet routinely collected in clinical practice. Many hospitals, however, do collect and store Formalin Fixed Paraffin Embedded (FFPE) tumor samples. Consequently, the sample size of whole genome cancer cohort studies could be increased tremendously by including FFPE samples, although the presence of artifacts might obfuscate the variant calling. To assess whether FFPE material can be used for cohort studies, we performed an in-depth comparison of somatic SNVs called on matching FF and FFPE Whole Genome Sequence (WGS) samples extracted from the same prostate metastatic tumor. Results. We first compared the calls between FF and FFPE, showing that on average 50% of the calls in FF are recovered in FFPE, with notable differences between variant callers. Remarkably, this overlap was better than the overlap between different variant callers on the same sample. Inspecting the Variant Allele Frequency (VAF), we observed that many of the calls common to FF and FFPE belonged to the same clonal subpopulation but were detected at a lower VAF in FFPE. We also demonstrated that these calls receive higher significance scores and are often identified by more than one variant caller. Based on this observation, we propose a simple heuristic to perform reliable variant calling in FFPE samples. Our heuristic identified 3684 common calls at a F1-score of 0.83. Conclusion. This study illustrates that when using the correct variant calling strategy, the overlap between the FF and FFPE sample in somatic SNVs increases to such an extent that a large fraction of the calls detected in the FFPE sample are contained in the FF sample and the number of variants unique to each sample remains restricted. These results suggest that somatic variants derived from WGS of FFPE material can be used in cohort studies.

scholarly journals Comparative analysis of somatic variant calling on matched FF and FFPE WGS samples

2020 ◽  
Author(s):  
Louise de Schaetzen van Brienen ◽  
Maarten Larmuseau ◽  
Kim Van der Eecken ◽  
Frederic De Ryck ◽  
Pauline Robbe ◽  
...  
Keyword(s):  
Cohort Studies ◽  
Variant Calling ◽  
Ffpe Sample ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Ffpe Tumor ◽  
Ffpe Samples ◽  
Fresh Frozen ◽  
Ffpe Material ◽  
The Individual

Abstract Background. Research grade Fresh Frozen (FF) DNA material is not yet routinely collected in clinical practice. Many hospitals, however, collect and store Formalin Fixed Paraffin Embedded (FFPE) tumor samples. Consequently, the sample size of whole genome cancer cohort studies could be increased tremendously by including FFPE samples, although the presence of artefacts might obfuscate the variant calling. To assess whether FFPE material can be used for cohort studies, we performed an in-depth comparison of somatic SNVs called on matching FF and FFPE Whole Genome Sequence (WGS) samples extracted from the same tumor. Results. We first compared the calls between an FF and an FFPE sample from a metastatic prostate tumor, showing that on average 50% of the calls in the FF are recovered in the FFPE sample, with notable differences between variant callers. Combining the variants of the different callers using a simple heuristic, increases both the precision and the sensitivity of the variant calling. Validating the heuristic on nine additional matched FF-FFPE samples, resulted in an average F1-score of 0.58 and an outperformance of any of the individual callers. In addition, we could show that part of the discrepancy between the FF and the FFPE samples can be attributed to intra-tumor heterogeneity (ITH). Conclusion. This study illustrates that when using the correct variant calling strategy, the majority of clonal SNVs can be recovered in an FFPE sample with high precision and sensitivity. These results suggest that somatic variants derived from WGS of FFPE material can be used in cohort studies.

scholarly journals Comparative analysis of somatic variant calling on matched FF and FFPE WGS samples

2020 ◽  
Author(s):  
Louise de Schaetzen van Brienen ◽  
Maarten Larmuseau ◽  
Kim Van der Eecken ◽  
Frederic De Ryck ◽  
Pauline Robbe ◽  
...  
Keyword(s):  
Cohort Studies ◽  
Variant Calling ◽  
Ffpe Sample ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Ffpe Tumor ◽  
Ffpe Samples ◽  
Fresh Frozen ◽  
Ffpe Material ◽  
The Individual

Abstract Background. Research grade Fresh Frozen (FF) DNA material is not yet routinely collected in clinical practice. Many hospitals, however, collect and store Formalin Fixed Paraffin Embedded (FFPE) tumor samples. Consequently, the sample size of whole genome cancer cohort studies could be increased tremendously by including FFPE samples, although the presence of artefacts might obfuscate the variant calling. To assess whether FFPE material can be used for cohort studies, we performed an in-depth comparison of somatic SNVs called on matching FF and FFPE Whole Genome Sequence (WGS) samples extracted from the same tumor. Results. We first compared the calls between an FF and an FFPE from a metastatic prostate tumor, showing that on average 50% of the calls in the FF are recovered in the FFPE sample, with notable differences between variant callers. Combining the variants of the different callers using a simple heuristic increases both the precision and the sensitivity of the variant calling. Validating the heuristic on nine additional matched FF-FFPE samples, resulted in an average F1-score of 0.58 and an outperformance of any of the individual callers. In addition, we could show that part of the discrepancy between the FF and the FFPE samples can be attributed to intra-tumor heterogeneity (ITH). Conclusion. This study illustrates that when using the correct variant calling strategy, the majority of clonal SNVs can be recovered in an FFPE sample with high precision and sensitivity. These results suggest that somatic variants derived from WGS of FFPE material can be used in cohort studies.

Extraction of DNA from a Wide Range of Objects by Treatment with Ammonium Salts

2020 ◽  
Vol 36 (6) ◽  
pp. 98-106
Author(s):  
E.I. Levitin ◽  
B.V. Sviridov ◽  
O.V. Piksasova ◽  
T.E. Shustikova
Keyword(s):  
Dna Extraction ◽  
Dna Isolation ◽  
Tissue Samples ◽  
New Approach ◽  
Cell Wall Disruption ◽  
Wide Range ◽  
Low Salt ◽  
Mammalian Blood ◽  
Plasmid Extraction ◽  
Two Hybrid

Currently, simple, rapid, and efficient techniques for DNA isolation from a wide range of organisms are in demand in biotechnology and bioinformatics. A key (and often limiting) step is the cell wall disruption and subsequent DNA extraction from the disintegrated cells. We have developed a new approach to DNA isolation from organisms with robust cell walls. The protocol includes the following steps: treatment of cells or tissue samples with ammonium acetate followed by cell lysis in low-salt buffer with the addition of SDS. Further DNA extraction is carried out according to standard methods. This approach is efficient for high-molecular native DNA isolation from bacteria, ascomycetes, yeast, and mammalian blood; it is also useful for express analysis of environmental microbial isolates and for plasmid extraction for two-hybrid library screening. express method for DNA isolation; ammonium salt treatment (в русских ключевых такой порядок), osmotic breakage of cells This study was financially supported by the NRC "Kurchatov Institute"-GOSNIIGENETIKA Kurchatov Genomic Center.

Sign in / Sign up

Export Citation Format

Share Document