scholarly journals Unlocking inaccessible historical genomes preserved in formalin

2021 ◽  
Author(s):  
Erin E. Hahn ◽  
Marina R. Alexander ◽  
Alicia Grealy ◽  
Jiri Stiller ◽  
Donald M. Gardiner ◽  
...  
Keyword(s):  
Temporal Trends ◽  
Nuclear Genome ◽  
Genomic Analysis ◽  
Proteinase K ◽  
Significant Advance ◽  
Alkaline Lysis ◽  
Dna Yield ◽  
Target Capture ◽  
Endogenous Sequence ◽  
Formalin Fixed

AbstractBackgroundMuseum specimens represent an unparalleled record of historical genomic data. However, the wide-spread practice of formalin preservation has thus far impeded genomic analysis of a large proportion of specimens. Limited DNA sequencing from formalin-preserved specimens has yielded low genomic coverage with unpredictable success. We set out to refine sample processing methods and to identify specimen characteristics predictive of sequencing success. With a set of taxonomically diverse specimens collected between 1936 and 2015 and ranging in preservation quality, we compared the efficacy of several end-to-end whole genome sequencing workflows alongside a k-mer-based trimming-free read alignment approach to maximize mapping of endogenous sequence.ResultsWe recovered complete mitochondrial genomes and up to 3X nuclear genome coverage from formalin-fixed tissues. Hot alkaline lysis coupled with phenol-chloroform extraction out- performed proteinase K digestion in recovering DNA, while library preparation method had little impact on sequencing success. The strongest predictor of DNA yield was overall specimen condition, which additively interacts with preservation conditions to accelerate DNA degradation.ConclusionsWe demonstrate a significant advance in capability beyond limited recovery of a small number of loci via PCR or target-capture sequencing. To facilitate strategic selection of suitable specimens for genomic sequencing, we present a decision-making framework that utilizes independent and non-destructive assessment criteria. Sequencing of formalin-fixed specimens will contribute to a greater understanding of temporal trends in genetic adaptation, including those associated with a changing climate. Our work enhances the value of museum collections worldwide by unlocking genomes of specimens that have been disregarded as a valid molecular resource.

scholarly journals Effect of Different Proteinase K Digest Protocols and Deparaffinization Methods on Yield and Integrity of DNA Extracted From Formalin-fixed, Paraffin-embedded Tissue

2020 ◽  
Vol 68 (3) ◽  
pp. 171-184 ◽  
Author(s):  
Zoe Frazer ◽  
Changyoung Yoo ◽  
Manveer Sroya ◽  
Camille Bellora ◽  
Brian L. DeWitt ◽  
...  
Keyword(s):  
Cell Number ◽  
Proteinase K ◽  
Dna Integrity ◽  
Epithelial Tumor ◽  
Dna Yield ◽  
Tissue Sections ◽  
Epithelial Tumor Cell ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

DNA extracted from formalin-fixed, paraffin-embedded tissue sections is often inadequate for sequencing, due to poor yield or degradation. We optimized the proteinase K digest by testing increased volume of enzyme and increased digest length from the manufacturer’s protocol using 54 biospecimens, performing the digest in centrifuge tubes. Doubling the quantity of proteinase K resulted in a median increase in yield of 96%. Applying the optimized proteinase K protocol to sections deparaffinized on microscope slides generated a further increase in yield of 41%, but only at >50,000 epithelial tumor cells/section. DNA yield now correlated with (χ2 = 0.84) and could be predicted from the epithelial tumor cell number. DNA integrity was assayed using end point multiplex PCR (amplicons of 100–400 bp visualized on a gel), quantitative PCR (qPCR; Illumina FFPE QC Assay), and nanoelectrophoresis (DNA Integrity Numbers [DINs]). Generally, increases in yield were accompanied by increases in integrity, but sometimes qPCR and DIN results were conflicting. Amplicons of 400 bp were almost universally obtained. The process of optimization enabled us to reduce the percentage of samples that failed published quality control thresholds for determining amenability to whole genome sequencing from 33% to 7%.

scholarly journals Assessing Quality and Functionality of DNA from Fresh and Archival Dried Blood Spots and Recommendations for Quality Control Guidelines

2007 ◽  
Vol 53 (8) ◽  
pp. 1401-1407 ◽  
Author(s):  
Malin Ida Linnea Sjöholm ◽  
Joakim Dillner ◽  
Joyce Carlson
Keyword(s):  
Dna Extraction ◽  
Multiple Displacement Amplification ◽  
Dried Blood Spots ◽  
Extraction Methods ◽  
Population Based ◽  
Alkaline Lysis ◽  
Screening Programs ◽  
Blood Spots ◽  
Dna Yield ◽  
Chelex 100

Abstract Background: Dried blood spots (DBS) are a convenient and inexpensive method for biobanking. Although many countries have established population-based DBS biobanks from neonatal screening programs, the quality and usefulness of DNA from DBS have not been extensively assessed. Methods: We compared 4 common DNA extraction methods (Qiagen, EZNA, Chelex 100, and alkaline lysis) in a pilot study using fresh DBS with known lymphocyte count. We assessed suitability for multiple displacement amplification (MDA) and subsequent single-nucleotide polymorphism (SNP) analyses. We selected the EZNA method for DNA extraction from archival samples up to 27 years old, stored at room temperature or −20 °C, and SNP analyses were performed after MDA. Results: Extraction using alkaline lysis failed in most tests, and Chelex 100 was unsuccessful in real-time PCR, whereas the EZNA and Qiagen methods were successful by all evaluated quality indices. DNA extraction by EZNA, MDA, and SNP analyses were successful for the archival samples stored at −20 °C. Conclusion: Routine protocols for evaluation of the quality and functional integrity of DNA based on DNA yield, DNA size, and quantification of amplifiable DNA allow use of sufficient template for MDA and successful SNP analyses from both primary DBS extract and MDA product. A single 3-mm disc can yield sufficient DNA for several thousand SNP analyses. DNA from DBS is thus suitable for genetic epidemiology studies.

scholarly journals Clinical and genetic diagnosis of thirteen Japanese patients with hereditary spherocytosis

2022 ◽  
Vol 9 (1) ◽  
Author(s):  
Keiko Shimojima Yamamoto ◽  
Taiju Utshigisawa ◽  
Hiromi Ogura ◽  
Takako Aoki ◽  
Takahiro Kawakami ◽  
...  
Keyword(s):  
Hemolytic Anemia ◽  
Hereditary Spherocytosis ◽  
Genetic Diagnosis ◽  
Genomic Analysis ◽  
Japanese Patients ◽  
Asian Countries ◽  
Target Capture ◽  
Detailed Family History ◽  
Target Capture Sequencing ◽  
Chronic Hemolytic Anemia

AbstractHereditary spherocytosis is the most frequent cause of hereditary hemolytic anemia and is classified into five subtypes (SPH1-5) according to OMIM. Because the clinical and laboratory features of patients with SPH1-5 are variable, it is difficult to classify these patients into the five subtypes based only on these features. We performed target capture sequencing in 51 patients with hemolytic anemia associated with/without morphological abnormalities in red blood cells. Thirteen variants were identified in five hereditary spherocytosis-related genes (six in ANK1 [SPH1]; four in SPTB [SPH2]; and one in each of SPTA1 [SPH3], SLC4A1 [SPH4], and EPB42 [SPH5]). Among these variants, seven were novel. The distribution pattern of the variants was different from that reported previously in Japan but similar to those reported in other Asian countries. Comprehensive genomic analysis would be useful and recommended, especially for patients without a detailed family history and those receiving frequent blood transfusions due to chronic hemolytic anemia.

Sodium Alginate versus Plasma Thrombin Cell Blocks in Diagnostic Cytopathology: A Comparative Analysis

2021 ◽  
pp. 1-7
Author(s):  
Shruti Gupta ◽  
Upasana Gautam ◽  
Shaily Susheilia ◽  
Baneet Bansal ◽  
Radha Uppal ◽  
...  
Keyword(s):  
Sodium Alginate ◽  
Molecular Analysis ◽  
Low Cost ◽  
Nuclear Marker ◽  
Fine Needle Aspirates ◽  
Dna Yield ◽  
Morphological Evaluation ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

<b><i>Background:</i></b> Cell blocks (CBs) are an essential adjunct in cytopathology practice. The aim of this study was to compare 2 techniques of CB preparation – plasma thrombin (PT) method with sodium alginate (SA) method for overall cellularity, morphological preservation, obscuring artefacts, immunocytochemistry (ICC), suitability for molecular analysis, and cost of preparation. <b><i>Design:</i></b> A total of 80 fine-needle aspirates from various sites and serous effusion samples were included. Of these cases, by random selection, 40 each were prepared by PT method and SA methods, respectively. The haematoxylin-eosin-stained sections from the formalin-fixed, paraffin-embedded CBs from both methods were evaluated in a blinded fashion by 2 cytopathologists and scored for cellularity, artefacts, and morphological preservation and analysed by χ<sup>2</sup> test with Yates correction. We evaluated 6 cases from each method by ICC for a range of membrane, cytoplasmic and nuclear marker expression. DNA was extracted from four cases to evaluate their utility for molecular analysis. <b><i>Results:</i></b> CB sections from PT and SA techniques showed comparable cellularity and excellent cytomorphological preservation. Blue gel-like artefacts were common in the SA technique but did not interfere with morphological evaluation. ICC staining results were also similar. DNA yield and utility for PCR were also comparable. The SA-CB cost half that of PT-CB (USD 0.4 vs. USD 1). <b><i>Conclusion:</i></b> SA technique of CB preparation is an excellent low-cost alternative to PT method for CB preparation.

scholarly journals Sequencing of Dna Isolated From Colorectal Sample Fixed in Liquid Formalin : Obstacles and Required Modifications

2020 ◽  
Vol 29 (2) ◽  
pp. 165-174
Author(s):  
Nahid Parvez ◽  
Mustak Ibn Ayub
Keyword(s):  
Dna Isolation ◽  
Low Cost ◽  
General Purpose ◽  
Proteinase K ◽  
Fixed Tissue ◽  
Target Region ◽  
Formalin Fixed Tissue ◽  
Successful Amplification ◽  
Cancer Tissues ◽  
Formalin Fixed

The necessary modifications in the protocol of general purpose DNA isolation kit to isolate and amplify a target region of genome from colorectal cancer tissues fixed in liquid formalin were made. It is shown that a one hour digestion with proteinase K yields enough DNA from formalin fixed colorectal tissue for subsequent PCR and sequencing. Moreover, using 100% ethanol instead of standard 50% during DNA binding step in the column improves the yield. As DNA fragmentation is unavoidable in formalin fixed tissue, PCR protocol was modified by increasing polymerase concentration to get successful amplification. Following these modifications, two regions of KRAS and BRAF genes were amplified and successfully sequenced from three different patients. These modifications provide a low cost option for Sanger sequencing of DNA isolated from formalin fixed tissue. Dhaka Univ. J. Biol. Sci. 29(2): 165-174, 2020 (July)

scholarly journals Maximizing RNA Yield from Archival Renal Tumors and Optimizing Gene Expression Analysis

2009 ◽  
Vol 15 (1) ◽  
pp. 80-85 ◽  
Author(s):  
Sean T. Glenn ◽  
Karen L. Head ◽  
Bin T. Teh ◽  
Kenneth W. Gross ◽  
Hyung L. Kim
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Reverse Transcription ◽  
Gene Expression Analysis ◽  
Rna Isolation ◽  
Proteinase K ◽  
Specific Primers ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Formalin-fixed, paraffin-embedded tissues are widely available for gene expression analysis using TaqMan ® PCR. Five methods, including 4 commercial kits, for recovering RNA from paraffin-embedded renal tumor tissue were compared. The MasterPure™ kit from Epicentre produced the highest RNA yield. However, the difference in RNA yield between the kit from Epicenter and Invitrogen’s TRIzol method was not significant. Using the top 3 RNA isolation methods, the manufacturers’ protocols were modified to include an overnight Proteinase K digestion. Overnight protein digestion resulted in a significant increase in RNA yield. To optimize the reverse transcription reaction, conventional reverse transcription with random oligonucleotide primers was compared to reverse transcription using primers specific for genes of interest. Reverse transcription using gene-specific primers significantly increased the quantity of cDNA detectable by TaqMan ® PCR. Therefore, expression profiling of formalin-fixed, paraffin-embedded tissue using TaqMan® qPCR can be optimized by using the MasterPure™ RNA isolation kit modified to include an overnight Proteinase K digestion and gene-specific primers during the reverse transcription.

scholarly journals How Do We Define the Genomic Landscape?

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. SCI-13-SCI-13
Author(s):  
Sandeep S. Dave
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Conflicts Of Interest ◽  
Genomic Analysis ◽  
Future Directions ◽  
Tissue Samples ◽  
Genomic Landscape ◽  
Fresh Frozen ◽  
Definition Of ◽  
Formalin Fixed

High throughput sequencing is a revolutionary technology for the definition of the genomic features of tumors. This talk will provide a review of the relevant methodologies for non-experts in the field. The presentation will include a discussion of how high throughput sequencing is performed, its relative strengths and weaknesses, and how it is applicable to formalin-fixed and fresh/frozen tissue samples. The talk will also describe future directions in the genomic analysis of tumors. Disclosures No relevant conflicts of interest to declare.

scholarly journals A protocol for good quality genomic DNA isolation from formalin-fixed paraffin-embedded tissues without using commercial kits

2021 ◽  
Author(s):  
Fazlur Rahman Talukdar ◽  
Irena Abramovic ◽  
Cyrille Cuenin ◽  
Christine Carreira ◽  
Nitin Gangane ◽  
...  
Keyword(s):  
Dna Isolation ◽  
Middle Income ◽  
Dna Yield ◽  
Genomic Dna Isolation ◽  
Novel Approach ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Commercial Kits ◽  
Formalin Fixed

DNA isolation from formalin fixed paraffin embedded (FFPE) tissues for molecular analysis has become a frequent procedure in cancer research. However, the yield or quality of the isolated DNA is often compromised, and commercial kits are used to overcome this to some extent. We developed a new protocol (IARCp) to improve better quality and yield of DNA from FFPE tissues without using any commercial kit. To evaluate the IARCp performance, we compared the quality and yield of DNA with two commercial kits, namely NucleoSpin DNA FFPE XS (MN) and QIAamp DNA Micro (QG) isolation kit. Total DNA yield for QG ranged from 120.0 to 282.0 ng (mean 216.5 ng), for MN: 213.6 to 394.2 ng (mean 319.1 ng), and with IARCp the yield was much higher ranging from 775.5 to 1896.9 ng (mean 1517.8 ng). Moreover, IARCp has also performed well in qualitative assessments. Overall, IARCp represents a novel approach to DNA isolation from FFPE which results in good quality and significant amounts of DNA suitable for many downstream genome-wide and targeted molecular analyses. Our proposed protocol does not require the use of any commercial kits for isolating DNA from FFPE tissues, making it suitable to implement in low-resource settings such as low and middle-income countries (LMICs).

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