Bias in RNA-seq Library Preparation: Current Challenges and Solutions

Figure 1

Simplified protocol of RNA-seq experiment and sources of bias. (a) Sample preservation and isolation. These biases can include sample degradation, DNA contamination. (b) Strategies for cDNA library construction. ①: the RNA directly converts to cDNA; then, cDNA was fragmented and library preparation. ②: classical a protocol. One method involves reverse transcription (RT) using random primers first, subsequently adapter ligations and sequencing (left). The other method is to first sequentially ligate 3 and 5 adapters, followed by performing cDNA synthesis with a primer complementary to the adapter (RT-primer), subsequently sequencing (right). On using the RT primer with a specific sequence, mispriming could occur due to annealing of the RT-primer to transcript sequences with some complementarity (RT mispriming). (c) RNA-seq platform (including Pyrosequencing, sequencing-by-synthesis, and single-molecule sequencing). These biases can be introduced by insertions and deletions, raw single-pass data, etc.

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