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BMC Genomics

, 18:442

Transcriptomic methods

Abstract

BackgroundRNA-sequencing RNA-seq has emerged as one of the most sensitive tool for gene expression analysis. Among the library preparation methods available, the standard polyA + enrichment provides a comprehensive, detailed, and accurate view of polyadenylated RNAs. However, on samples of suboptimal quality ribosomal RNA depletion and exon capture methods have recently been reported as better alternatives.

MethodsWe compared for the first time three commercial Illumina library preparation kits TruSeq Stranded mRNA, TruSeq Ribo-Zero rRNA Removal, and TruSeq RNA Access as representatives of these three different approaches using well-established human reference RNA samples from the MAQC-SEQC consortium on a wide range of input amounts from 100 ng down to 1 ng and degradation levels intact, degraded, and highly degraded.

ResultsWe assessed the accuracy of the generated expression values by comparison to gold standard TaqMan qPCR measurements and gained unprecedented insight into the limits of applicability in terms of input quantity and sample quality of each protocol. We found that each protocol generates highly reproducible results R > 0.92 on intact RNA samples down to input amounts of 10 ng. For degraded RNA samples, Ribo-Zero showed clear performance advantages over the other two protocols as it generated more accurate and better reproducible gene expression results even at very low input amounts such as 1 ng and 2 ng. For highly degraded RNA samples, RNA Access performed best generating reliable data down to 5 ng input.

ConclusionsWe found that the ribosomal RNA depletion protocol from Illumina works very well at amounts far below recommendation and over a good range of intact and degraded material. We also infer that the exome-capture protocol RNA Access, Illumina performs better than other methods on highly degraded and low amount samples.

KeywordsRNA-sequencing Expression profiling Benchmarking Low quality Low quantity Differential expression AbbreviationMAQC-SEQCMicroarray-Sequencing Quality Control

Electronic supplementary materialThe online version of this article doi:10.1186-s12864-017-3827-y contains supplementary material, which is available to authorized users.





Autor: Sven Schuierer - Walter Carbone - Judith Knehr - Virginie Petitjean - Anita Fernandez - Marc Sultan - Guglielmo Roma

Fuente: https://link.springer.com/



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