A comparative analysis of library prep approaches for sequencing low input translatome samples
Abstract Background Cell type-specific ribosome-pulldown has become an increasingly popular method for analysis of gene expression. It allows for expression analysis from intact tissues and monitoring of protein synthesis in vivo. However, while its utility has been assessed, technical aspects relat...
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BMC
2018-09-01
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Series: | BMC Genomics |
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Online Access: | http://link.springer.com/article/10.1186/s12864-018-5066-2 |
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author | Yang Song Beatrice Milon Sandra Ott Xuechu Zhao Lisa Sadzewicz Amol Shetty Erich T. Boger Luke J. Tallon Robert J. Morell Anup Mahurkar Ronna Hertzano |
author_facet | Yang Song Beatrice Milon Sandra Ott Xuechu Zhao Lisa Sadzewicz Amol Shetty Erich T. Boger Luke J. Tallon Robert J. Morell Anup Mahurkar Ronna Hertzano |
author_sort | Yang Song |
collection | DOAJ |
description | Abstract Background Cell type-specific ribosome-pulldown has become an increasingly popular method for analysis of gene expression. It allows for expression analysis from intact tissues and monitoring of protein synthesis in vivo. However, while its utility has been assessed, technical aspects related to sequencing of these samples, often starting with a smaller amount of RNA, have not been reported. In this study, we evaluated the performance of five library prep protocols for ribosome-associated mRNAs when only 250 pg-4 ng of total RNA are used. Results We obtained total and RiboTag-IP RNA, in three biological replicates. We compared 5 methods of library preparation for Illumina Next Generation sequencing: NuGEN Ovation RNA-Seq system V2 Kit, TaKaRa SMARTer Stranded Total RNA-Seq Kit, TaKaRa SMART-Seq v4 Ultra Low Input RNA Kit, Illumina TruSeq RNA Library Prep Kit v2 and NEBNext® Ultra™ Directional RNA Library Prep Kit using slightly modified protocols each with 4 ng of total RNA. An additional set of samples was processed using the TruSeq kit with 70 ng, as a ‘gold standard’ control and the SMART-Seq v4 with 250 pg of total RNA. TruSeq-processed samples had the best metrics overall, with similar results for the 4 ng and 70 ng samples. The results of the SMART-Seq v4 processed samples were similar to TruSeq (Spearman correlation > 0.8) despite using lower amount of input RNA. All RiboTag-IP samples had an increase in the intronic reads compared with the corresponding whole tissue, suggesting that the IP captures some immature mRNAs. The SMARTer-processed samples had a higher representation of ribosomal and non-coding RNAs leading to lower representation of protein coding mRNA. The enrichment or depletion of IP samples compared to corresponding input RNA was similar across all kits except for SMARTer kit. Conclusion RiboTag-seq can be performed successfully with as little as 250 pg of total RNA when using the SMART-Seq v4 kit and 4 ng when using the modified protocols of other library preparation kits. The SMART-Seq v4 and TruSeq kits resulted in the highest quality libraries. RiboTag IP RNA contains some immature transcripts. |
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spelling | doaj.art-ded22db5c2154d028ffb1492693fae892022-12-22T00:50:07ZengBMCBMC Genomics1471-21642018-09-0119111610.1186/s12864-018-5066-2A comparative analysis of library prep approaches for sequencing low input translatome samplesYang Song0Beatrice Milon1Sandra Ott2Xuechu Zhao3Lisa Sadzewicz4Amol Shetty5Erich T. Boger6Luke J. Tallon7Robert J. Morell8Anup Mahurkar9Ronna Hertzano10Institute for Genome Sciences, University of Maryland School of MedicineDepartment of Otorhinolaryngology-Head & Neck Surgery, University of Maryland School of MedicineInstitute for Genome Sciences, University of Maryland School of MedicineInstitute for Genome Sciences, University of Maryland School of MedicineInstitute for Genome Sciences, University of Maryland School of MedicineInstitute for Genome Sciences, University of Maryland School of MedicineGenomics and Computational Biology Core, National Institute on Deafness and Other Communication DisordersInstitute for Genome Sciences, University of Maryland School of MedicineGenomics and Computational Biology Core, National Institute on Deafness and Other Communication DisordersInstitute for Genome Sciences, University of Maryland School of MedicineInstitute for Genome Sciences, University of Maryland School of MedicineAbstract Background Cell type-specific ribosome-pulldown has become an increasingly popular method for analysis of gene expression. It allows for expression analysis from intact tissues and monitoring of protein synthesis in vivo. However, while its utility has been assessed, technical aspects related to sequencing of these samples, often starting with a smaller amount of RNA, have not been reported. In this study, we evaluated the performance of five library prep protocols for ribosome-associated mRNAs when only 250 pg-4 ng of total RNA are used. Results We obtained total and RiboTag-IP RNA, in three biological replicates. We compared 5 methods of library preparation for Illumina Next Generation sequencing: NuGEN Ovation RNA-Seq system V2 Kit, TaKaRa SMARTer Stranded Total RNA-Seq Kit, TaKaRa SMART-Seq v4 Ultra Low Input RNA Kit, Illumina TruSeq RNA Library Prep Kit v2 and NEBNext® Ultra™ Directional RNA Library Prep Kit using slightly modified protocols each with 4 ng of total RNA. An additional set of samples was processed using the TruSeq kit with 70 ng, as a ‘gold standard’ control and the SMART-Seq v4 with 250 pg of total RNA. TruSeq-processed samples had the best metrics overall, with similar results for the 4 ng and 70 ng samples. The results of the SMART-Seq v4 processed samples were similar to TruSeq (Spearman correlation > 0.8) despite using lower amount of input RNA. All RiboTag-IP samples had an increase in the intronic reads compared with the corresponding whole tissue, suggesting that the IP captures some immature mRNAs. The SMARTer-processed samples had a higher representation of ribosomal and non-coding RNAs leading to lower representation of protein coding mRNA. The enrichment or depletion of IP samples compared to corresponding input RNA was similar across all kits except for SMARTer kit. Conclusion RiboTag-seq can be performed successfully with as little as 250 pg of total RNA when using the SMART-Seq v4 kit and 4 ng when using the modified protocols of other library preparation kits. The SMART-Seq v4 and TruSeq kits resulted in the highest quality libraries. RiboTag IP RNA contains some immature transcripts.http://link.springer.com/article/10.1186/s12864-018-5066-2RiboTagLibrary preparation kitsLow-input RNA-seqRNA-seqCoverage bias |
spellingShingle | Yang Song Beatrice Milon Sandra Ott Xuechu Zhao Lisa Sadzewicz Amol Shetty Erich T. Boger Luke J. Tallon Robert J. Morell Anup Mahurkar Ronna Hertzano A comparative analysis of library prep approaches for sequencing low input translatome samples BMC Genomics RiboTag Library preparation kits Low-input RNA-seq RNA-seq Coverage bias |
title | A comparative analysis of library prep approaches for sequencing low input translatome samples |
title_full | A comparative analysis of library prep approaches for sequencing low input translatome samples |
title_fullStr | A comparative analysis of library prep approaches for sequencing low input translatome samples |
title_full_unstemmed | A comparative analysis of library prep approaches for sequencing low input translatome samples |
title_short | A comparative analysis of library prep approaches for sequencing low input translatome samples |
title_sort | comparative analysis of library prep approaches for sequencing low input translatome samples |
topic | RiboTag Library preparation kits Low-input RNA-seq RNA-seq Coverage bias |
url | http://link.springer.com/article/10.1186/s12864-018-5066-2 |
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