Application of the 3′ mRNA-Seq using unique molecular identifiers in highly degraded RNA derived from formalin-fixed, paraffin-embedded tissue
Abstract Background Archival formalin-fixed, paraffin-embedded (FFPE) tissue samples with clinical and histological data are a singularly valuable resource for developing new molecular biomarkers. However, transcriptome analysis remains challenging with standard mRNA-seq methods as FFPE derived-RNA...
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| Format: | Article |
| Language: | English |
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BMC
2021-10-01
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| Series: | BMC Genomics |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12864-021-08068-1 |
| _version_ | 1818897038471856128 |
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| author | Jin Sung Jang Eileen Holicky Julie Lau Samantha McDonough Mark Mutawe Matthew J. Koster Kenneth J. Warrington Julie M. Cuninngham |
| author_facet | Jin Sung Jang Eileen Holicky Julie Lau Samantha McDonough Mark Mutawe Matthew J. Koster Kenneth J. Warrington Julie M. Cuninngham |
| author_sort | Jin Sung Jang |
| collection | DOAJ |
| description | Abstract Background Archival formalin-fixed, paraffin-embedded (FFPE) tissue samples with clinical and histological data are a singularly valuable resource for developing new molecular biomarkers. However, transcriptome analysis remains challenging with standard mRNA-seq methods as FFPE derived-RNA samples are often highly modified and fragmented. The recently developed 3′ mRNA-seq method sequences the 3′ region of mRNA using unique molecular identifiers (UMI), thus generating gene expression data with minimal PCR bias. In this study, we evaluated the performance of 3′ mRNA-Seq using Lexogen QuantSeq 3′ mRNA-Seq Library Prep Kit FWD with UMI, comparing with TruSeq Stranded mRNA-Seq and RNA Exome Capture kit. The fresh-frozen (FF) and FFPE tissues yielded nucleotide sizes range from 13 to > 70% of DV200 values; input amounts ranged from 1 ng to 100 ng for validation. Results The total mapped reads of QuantSeq 3′ mRNA-Seq to the reference genome ranged from 99 to 74% across all samples. After PCR bias correction, 3 to 56% of total sequenced reads were retained. QuantSeq 3′ mRNA-Seq data showed highly reproducible data across replicates in Universal Human Reference RNA (UHR, R > 0.94) at input amounts from 1 ng to 100 ng, and FF and FFPE paired samples (R = 0.92) at 10 ng. Severely degraded FFPE RNA with ≤30% of DV200 value showed good concordance (R > 0.87) with 100 ng input. A moderate correlation was observed when directly comparing QuantSeq 3′ mRNA-Seq data with TruSeq Stranded mRNA-Seq (R = 0.78) and RNA Exome Capture data (R > 0.67). Conclusion In this study, QuantSeq 3′ mRNA-Seq with PCR bias correction using UMI is shown to be a suitable method for gene quantification in both FF and FFPE RNAs. 3′ mRNA-Seq with UMI may be applied to severely degraded RNA from FFPE tissues generating high-quality sequencing data. |
| first_indexed | 2024-12-19T19:09:49Z |
| format | Article |
| id | doaj.art-d18cfbe9b1934138adbf514a838ede3a |
| institution | Directory Open Access Journal |
| issn | 1471-2164 |
| language | English |
| last_indexed | 2024-12-19T19:09:49Z |
| publishDate | 2021-10-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Genomics |
| spelling | doaj.art-d18cfbe9b1934138adbf514a838ede3a2022-12-21T20:09:20ZengBMCBMC Genomics1471-21642021-10-012211910.1186/s12864-021-08068-1Application of the 3′ mRNA-Seq using unique molecular identifiers in highly degraded RNA derived from formalin-fixed, paraffin-embedded tissueJin Sung Jang0Eileen Holicky1Julie Lau2Samantha McDonough3Mark Mutawe4Matthew J. Koster5Kenneth J. Warrington6Julie M. Cuninngham7Genome Analysis Core, Medical Genome Facility, Center for Individualized Medicine, Mayo ClinicGenome Analysis Core, Medical Genome Facility, Center for Individualized Medicine, Mayo ClinicGenome Analysis Core, Medical Genome Facility, Center for Individualized Medicine, Mayo ClinicGenome Analysis Core, Medical Genome Facility, Center for Individualized Medicine, Mayo ClinicGenome Analysis Core, Medical Genome Facility, Center for Individualized Medicine, Mayo ClinicDepartment of Internal Medicine, Division of Rheumatology, Mayo ClinicDepartment of Internal Medicine, Division of Rheumatology, Mayo ClinicGenome Analysis Core, Medical Genome Facility, Center for Individualized Medicine, Mayo ClinicAbstract Background Archival formalin-fixed, paraffin-embedded (FFPE) tissue samples with clinical and histological data are a singularly valuable resource for developing new molecular biomarkers. However, transcriptome analysis remains challenging with standard mRNA-seq methods as FFPE derived-RNA samples are often highly modified and fragmented. The recently developed 3′ mRNA-seq method sequences the 3′ region of mRNA using unique molecular identifiers (UMI), thus generating gene expression data with minimal PCR bias. In this study, we evaluated the performance of 3′ mRNA-Seq using Lexogen QuantSeq 3′ mRNA-Seq Library Prep Kit FWD with UMI, comparing with TruSeq Stranded mRNA-Seq and RNA Exome Capture kit. The fresh-frozen (FF) and FFPE tissues yielded nucleotide sizes range from 13 to > 70% of DV200 values; input amounts ranged from 1 ng to 100 ng for validation. Results The total mapped reads of QuantSeq 3′ mRNA-Seq to the reference genome ranged from 99 to 74% across all samples. After PCR bias correction, 3 to 56% of total sequenced reads were retained. QuantSeq 3′ mRNA-Seq data showed highly reproducible data across replicates in Universal Human Reference RNA (UHR, R > 0.94) at input amounts from 1 ng to 100 ng, and FF and FFPE paired samples (R = 0.92) at 10 ng. Severely degraded FFPE RNA with ≤30% of DV200 value showed good concordance (R > 0.87) with 100 ng input. A moderate correlation was observed when directly comparing QuantSeq 3′ mRNA-Seq data with TruSeq Stranded mRNA-Seq (R = 0.78) and RNA Exome Capture data (R > 0.67). Conclusion In this study, QuantSeq 3′ mRNA-Seq with PCR bias correction using UMI is shown to be a suitable method for gene quantification in both FF and FFPE RNAs. 3′ mRNA-Seq with UMI may be applied to severely degraded RNA from FFPE tissues generating high-quality sequencing data.https://doi.org/10.1186/s12864-021-08068-13′ mRNA-SeqUMIFFPEPCR amplification biasGene expression |
| spellingShingle | Jin Sung Jang Eileen Holicky Julie Lau Samantha McDonough Mark Mutawe Matthew J. Koster Kenneth J. Warrington Julie M. Cuninngham Application of the 3′ mRNA-Seq using unique molecular identifiers in highly degraded RNA derived from formalin-fixed, paraffin-embedded tissue BMC Genomics 3′ mRNA-Seq UMI FFPE PCR amplification bias Gene expression |
| title | Application of the 3′ mRNA-Seq using unique molecular identifiers in highly degraded RNA derived from formalin-fixed, paraffin-embedded tissue |
| title_full | Application of the 3′ mRNA-Seq using unique molecular identifiers in highly degraded RNA derived from formalin-fixed, paraffin-embedded tissue |
| title_fullStr | Application of the 3′ mRNA-Seq using unique molecular identifiers in highly degraded RNA derived from formalin-fixed, paraffin-embedded tissue |
| title_full_unstemmed | Application of the 3′ mRNA-Seq using unique molecular identifiers in highly degraded RNA derived from formalin-fixed, paraffin-embedded tissue |
| title_short | Application of the 3′ mRNA-Seq using unique molecular identifiers in highly degraded RNA derived from formalin-fixed, paraffin-embedded tissue |
| title_sort | application of the 3 mrna seq using unique molecular identifiers in highly degraded rna derived from formalin fixed paraffin embedded tissue |
| topic | 3′ mRNA-Seq UMI FFPE PCR amplification bias Gene expression |
| url | https://doi.org/10.1186/s12864-021-08068-1 |
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