Artifacts and biases of the reverse transcription reaction in RNA sequencing.

RNA sequencing has spurred a significant number of research areas in recent years. Most protocols rely on synthesizing a more stable complementary DNA (cDNA) copy of the RNA molecule during the reverse transcription reaction. The resulting cDNA pool is often wrongfully assumed to be quantitatively and molecularly similar to the original RNA input. Sadly, biases and artifacts confound the resulting cDNA mixture. These issues are often overlooked or ignored in the literature by those that rely on the reverse transcription process. In this review, we confront the reader with intra- and intersample biases and artifacts caused by the reverse transcription reaction during RNA sequencing experiments. To fight the reader's despair, we also provide solutions to most issues and inform on good RNA sequencing practices. We hope the reader can use this review to their advantage, thereby contributing to scientifically sound RNA studies.

Blocking Abundant RNA Transcripts by High-Affinity Oligonucleotides during Transcriptome Library Preparation.

BACKGROUND: RNA sequencing has become the gold standard for transcriptome analysis but has an inherent limitation of challenging quantification of low-abundant transcripts. In contrast to microarray technology, RNA sequencing reads are proportionally divided in function of transcript abundance. Therefore, low-abundant RNAs compete against highly abundant - and sometimes non-informative - RNA species. RESULTS: We developed an easy-to-use strategy based on high-affinity RNA-binding oligonucleotides to block reverse transcription and PCR amplification of specific RNA transcripts, thereby substantially reducing their abundance in the final sequencing library. To demonstrate the broad application potential of our method, we applied it to different transcripts and library preparation strategies, including YRNAs in small RNA sequencing of human blood plasma, mitochondrial rRNAs in both 3' end sequencing and long-read sequencing, and MALAT1 in single-cell 3' end sequencing. We demonstrate that the blocking strategy is highly efficient, reproducible, specific, and generally results in better transcriptome coverage and complexity. CONCLUSION: Our method does not require modifications of the library preparation procedure apart from simply adding blocking oligonucleotides to the RT reaction and can thus be easily integrated into virtually any RNA sequencing library preparation protocol.

The Dangers of Using Cq to Quantify Nucleic Acid in Biological Samples: A Lesson From COVID-19.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA quantities, measured by reverse transcription quantitative PCR (RT-qPCR), have been proposed to stratify clinical risk or determine analytical performance targets. We investigated reproducibility and how setting diagnostic cutoffs altered the clinical sensitivity of coronavirus disease 2019 (COVID-19) testing. METHODS: Quantitative SARS-CoV-2 RNA distributions [quantification cycle (Cq) and copies/mL] from more than 6000 patients from 3 clinical laboratories in United Kingdom, Belgium, and the Republic of Korea were analyzed. Impact of Cq cutoffs on clinical sensitivity was assessed. The June/July 2020 INSTAND external quality assessment scheme SARS-CoV-2 materials were used to estimate laboratory reported copies/mL and to estimate the variation in copies/mL for a given Cq. RESULTS: When the WHO-suggested Cq cutoff of 25 was applied, the clinical sensitivity dropped to about 16%. Clinical sensitivity also dropped to about 27% when a simulated limit of detection of 106 copies/mL was applied. The interlaboratory variation for a given Cq value was >1000 fold in copies/mL (99% CI). CONCLUSION: While RT-qPCR has been instrumental in the response to COVID-19, we recommend Cq (cycle threshold or crossing point) values not be used to set clinical cutoffs or diagnostic performance targets due to poor interlaboratory reproducibility; calibrated copy-based units (used elsewhere in virology) offer more reproducible alternatives. We also report a phenomenon where diagnostic performance may change relative to the effective reproduction number. Our findings indicate that the disparities between patient populations across time are an important consideration when evaluating or deploying diagnostic tests. This is especially relevant to the emergency situation of an evolving pandemic.

Evaluation of efficiency and sensitivity of 1D and 2D sample pooling strategies for SARS-CoV-2 RT-qPCR screening purposes.

To increase the throughput, lower the cost, and save scarce test reagents, laboratories can pool patient samples before SARS-CoV-2 RT-qPCR testing. While different sample pooling methods have been proposed and effectively implemented in some laboratories, no systematic and large-scale evaluations exist using real-life quantitative data gathered throughout the different epidemiological stages. Here, we use anonymous data from 9673 positive cases to model, simulate and compare 1D and 2D pooling strategies. We show that the optimal choice of pooling method and pool size is an intricate decision with a testing population-dependent efficiency-sensitivity trade-off and present an online tool to provide the reader with custom real-time 1D pooling strategy recommendations.