دورية أكاديمية
أعرض في EDS

Quality control recommendations for RNASeq using FFPE samples based on pre-sequencing lab metrics and post-sequencing bioinformatics metrics.

التفاصيل البيبلوغرافية
العنوان: Quality control recommendations for RNASeq using FFPE samples based on pre-sequencing lab metrics and post-sequencing bioinformatics metrics.
المؤلفون: Liu Y; Department of Quantitative Health Sciences, Mayo Clinic, 200 1st Street SW, Rochester, MN, 55905, USA., Bhagwate A; Department of Quantitative Health Sciences, Mayo Clinic, 200 1st Street SW, Rochester, MN, 55905, USA., Winham SJ; Department of Quantitative Health Sciences, Mayo Clinic, 200 1st Street SW, Rochester, MN, 55905, USA., Stephens MT; Genomics and Bioinformatics Core Facility, 019 Galvin Life Sciences Center, University of Notre Dame, Notre Dame, IN, 46556, USA., Harker BW; Genomics and Bioinformatics Core Facility, 019 Galvin Life Sciences Center, University of Notre Dame, Notre Dame, IN, 46556, USA., McDonough SJ; Department of Laboratory Medicine and Pathology, Mayo Clinic, 200 1st Street SW, Rochester, MN, 55905, USA., Stallings-Mann ML; Department of Neuroscience, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL, 32224, USA., Heinzen EP; Department of Quantitative Health Sciences, Mayo Clinic, 200 1st Street SW, Rochester, MN, 55905, USA., Vierkant RA; Department of Quantitative Health Sciences, Mayo Clinic, 200 1st Street SW, Rochester, MN, 55905, USA., Hoskin TL; Department of Quantitative Health Sciences, Mayo Clinic, 200 1st Street SW, Rochester, MN, 55905, USA., Frost MH; Department of Medical Oncology, Mayo Clinic, 200 1st Street SW, Rochester, MN, 55905, USA., Carter JM; Department of Laboratory Medicine and Pathology, Mayo Clinic, 200 1st Street SW, Rochester, MN, 55905, USA., Pfrender ME; Department of Biological Sciences, 109B Galvin Life Science Center, University of Notre Dame, Notre Dame, IN, 46556, USA., Littlepage L; Department of Chemistry and Biochemistry, Harper Cancer Research Center, University of Notre Dame, Notre Dame, IN, 46556, USA., Radisky DC; Department of Cancer Biology, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL, 32224, USA., Cunningham JM; Department of Laboratory Medicine and Pathology, Mayo Clinic, 200 1st Street SW, Rochester, MN, 55905, USA., Degnim AC; Department of Surgery, Mayo Clinic, 200 1st Street SW, Rochester, MN, 55905, USA., Wang C; Department of Quantitative Health Sciences, Mayo Clinic, 200 1st Street SW, Rochester, MN, 55905, USA. wang.chen@mayo.edu.
المصدر: BMC medical genomics [BMC Med Genomics] 2022 Sep 16; Vol. 15 (1), pp. 195. Date of Electronic Publication: 2022 Sep 16.
نوع المنشور: Journal Article; Research Support, N.I.H., Extramural
اللغة: English
بيانات الدورية: Publisher: BioMed Central Country of Publication: England NLM ID: 101319628 Publication Model: Electronic Cited Medium: Internet ISSN: 1755-8794 (Electronic) Linking ISSN: 17558794 NLM ISO Abbreviation: BMC Med Genomics Subsets: MEDLINE
أسماء مطبوعة: Original Publication: London : BioMed Central
مواضيع طبية MeSH: Benchmarking* , Computational Biology*, Biomarkers ; Female ; Formaldehyde ; Humans ; Paraffin Embedding ; Quality Control ; RNA ; Sequence Analysis, RNA/methods ; Tissue Fixation
مستخلص: Background: Formalin-fixed, paraffin-embedded (FFPE) tissues have many advantages for identification of risk biomarkers, including wide availability and potential for extended follow-up endpoints. However, RNA derived from archival FFPE samples has limited quality. Here we identified parameters that determine which FFPE samples have the potential for successful RNA extraction, library preparation, and generation of usable RNAseq data.
Methods: We optimized library preparation protocols designed for use with FFPE samples using seven FFPE and Fresh Frozen replicate pairs, and tested optimized protocols using a study set of 130 FFPE biopsies from women with benign breast disease. Metrics from RNA extraction and preparation procedures were collected and compared with bioinformatics sequencing summary statistics. Finally, a decision tree model was built to learn the relationship between pre-sequencing lab metrics and qc pass/fail status as determined by bioinformatics metrics.
Results: Samples that failed bioinformatics qc tended to have low median sample-wise correlation within the cohort (Spearman correlation < 0.75), low number of reads mapped to gene regions (< 25 million), or low number of detectable genes (11,400 # of detected genes with TPM > 4). The median RNA concentration and pre-capture library Qubit values for qc failed samples were 18.9 ng/ul and 2.08 ng/ul respectively, which were significantly lower than those of qc pass samples (40.8 ng/ul and 5.82 ng/ul). We built a decision tree model based on input RNA concentration, input library qubit values, and achieved an F score of 0.848 in predicting QC status (pass/fail) of FFPE samples.
Conclusions: We provide a bioinformatics quality control recommendation for FFPE samples from breast tissue by evaluating bioinformatic and sample metrics. Our results suggest a minimum concentration of 25 ng/ul FFPE-extracted RNA for library preparation and 1.7 ng/ul pre-capture library output to achieve adequate RNA-seq data for downstream bioinformatics analysis.
(© 2022. The Author(s).)
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معلومات مُعتمدة: P30 CA015083 United States CA NCI NIH HHS; P50 CA116201 United States CA NCI NIH HHS; R01 CA187112 United States CA NCI NIH HHS
فهرسة مساهمة: Keywords: Breast tissue; DV200; DV50; Decision tree; FFPE; Library concentration; Quality control; RNA concentration; RNA-seq
المشرفين على المادة: 0 (Biomarkers)
1HG84L3525 (Formaldehyde)
63231-63-0 (RNA)
تواريخ الأحداث: Date Created: 20220916 Date Completed: 20220920 Latest Revision: 20221216
رمز التحديث: 20231215
مُعرف محوري في PubMed: PMC9479231
DOI: 10.1186/s12920-022-01355-0
PMID: 36114500
قاعدة البيانات: MEDLINE
الوصف
تدمد:1755-8794
DOI:10.1186/s12920-022-01355-0