The impact of RNA sequence library construction protocols on transcriptomic profiling of leukemia
Abstract Background RNA sequencing (RNA-seq) has become an indispensable tool to identify disease associated transcriptional profiles and determine the molecular underpinnings of diseases. However, the broad adaptation of the methodology into the clinic is still hampered by inconsistent results from...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2017-08-01
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| Series: | BMC Genomics |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12864-017-4039-1 |
| _version_ | 1831664818442993664 |
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| author | Ashwini Kumar Matti Kankainen Alun Parsons Olli Kallioniemi Pirkko Mattila Caroline A. Heckman |
| author_facet | Ashwini Kumar Matti Kankainen Alun Parsons Olli Kallioniemi Pirkko Mattila Caroline A. Heckman |
| author_sort | Ashwini Kumar |
| collection | DOAJ |
| description | Abstract Background RNA sequencing (RNA-seq) has become an indispensable tool to identify disease associated transcriptional profiles and determine the molecular underpinnings of diseases. However, the broad adaptation of the methodology into the clinic is still hampered by inconsistent results from different RNA-seq protocols and involves further evaluation of its analytical reliability using patient samples. Here, we applied two commonly used RNA-seq library preparation protocols to samples from acute leukemia patients to understand how poly-A-tailed mRNA selection (PA) and ribo-depletion (RD) based RNA-seq library preparation protocols affect gene fusion detection, variant calling, and gene expression profiling. Results Overall, the protocols produced similar results with consistent outcomes. Nevertheless, the PA protocol was more efficient in quantifying expression of leukemia marker genes and showed better performance in the expression-based classification of leukemia. Independent qRT-PCR experiments verified that the PA protocol better represented total RNA compared to the RD protocol. In contrast, the RD protocol detected a higher number of non-coding RNA features and had better alignment efficiency. The RD protocol also recovered more known fusion-gene events, although variability was seen in fusion gene predictions. Conclusion The overall findings provide a framework for the use of RNA-seq in a precision medicine setting with limited number of samples and suggest that selection of the library preparation protocol should be based on the objectives of the analysis. |
| first_indexed | 2024-12-19T19:22:18Z |
| format | Article |
| id | doaj.art-0ae52d828a7d4dfab89cc8e7d40cfb75 |
| institution | Directory Open Access Journal |
| issn | 1471-2164 |
| language | English |
| last_indexed | 2024-12-19T19:22:18Z |
| publishDate | 2017-08-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Genomics |
| spelling | doaj.art-0ae52d828a7d4dfab89cc8e7d40cfb752022-12-21T20:08:56ZengBMCBMC Genomics1471-21642017-08-0118111310.1186/s12864-017-4039-1The impact of RNA sequence library construction protocols on transcriptomic profiling of leukemiaAshwini Kumar0Matti Kankainen1Alun Parsons2Olli Kallioniemi3Pirkko Mattila4Caroline A. Heckman5Institute for Molecular Medicine Finland (FIMM), Helsinki Institute of Life Science, University of HelsinkiInstitute for Molecular Medicine Finland (FIMM), Helsinki Institute of Life Science, University of HelsinkiInstitute for Molecular Medicine Finland (FIMM), Helsinki Institute of Life Science, University of HelsinkiInstitute for Molecular Medicine Finland (FIMM), Helsinki Institute of Life Science, University of HelsinkiInstitute for Molecular Medicine Finland (FIMM), Helsinki Institute of Life Science, University of HelsinkiInstitute for Molecular Medicine Finland (FIMM), Helsinki Institute of Life Science, University of HelsinkiAbstract Background RNA sequencing (RNA-seq) has become an indispensable tool to identify disease associated transcriptional profiles and determine the molecular underpinnings of diseases. However, the broad adaptation of the methodology into the clinic is still hampered by inconsistent results from different RNA-seq protocols and involves further evaluation of its analytical reliability using patient samples. Here, we applied two commonly used RNA-seq library preparation protocols to samples from acute leukemia patients to understand how poly-A-tailed mRNA selection (PA) and ribo-depletion (RD) based RNA-seq library preparation protocols affect gene fusion detection, variant calling, and gene expression profiling. Results Overall, the protocols produced similar results with consistent outcomes. Nevertheless, the PA protocol was more efficient in quantifying expression of leukemia marker genes and showed better performance in the expression-based classification of leukemia. Independent qRT-PCR experiments verified that the PA protocol better represented total RNA compared to the RD protocol. In contrast, the RD protocol detected a higher number of non-coding RNA features and had better alignment efficiency. The RD protocol also recovered more known fusion-gene events, although variability was seen in fusion gene predictions. Conclusion The overall findings provide a framework for the use of RNA-seq in a precision medicine setting with limited number of samples and suggest that selection of the library preparation protocol should be based on the objectives of the analysis.http://link.springer.com/article/10.1186/s12864-017-4039-1RNA-sequencingHematological malignanciesLibrary preparationRibo-depletionPoly-A selection |
| spellingShingle | Ashwini Kumar Matti Kankainen Alun Parsons Olli Kallioniemi Pirkko Mattila Caroline A. Heckman The impact of RNA sequence library construction protocols on transcriptomic profiling of leukemia BMC Genomics RNA-sequencing Hematological malignancies Library preparation Ribo-depletion Poly-A selection |
| title | The impact of RNA sequence library construction protocols on transcriptomic profiling of leukemia |
| title_full | The impact of RNA sequence library construction protocols on transcriptomic profiling of leukemia |
| title_fullStr | The impact of RNA sequence library construction protocols on transcriptomic profiling of leukemia |
| title_full_unstemmed | The impact of RNA sequence library construction protocols on transcriptomic profiling of leukemia |
| title_short | The impact of RNA sequence library construction protocols on transcriptomic profiling of leukemia |
| title_sort | impact of rna sequence library construction protocols on transcriptomic profiling of leukemia |
| topic | RNA-sequencing Hematological malignancies Library preparation Ribo-depletion Poly-A selection |
| url | http://link.springer.com/article/10.1186/s12864-017-4039-1 |
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