Enhanced methods for unbiased deep sequencing of Lassa and Ebola RNA viruses from clinical and biological samples.
Bottom Line: This depletion step improves both the quality of data and quantity of informative reads in unbiased total RNA sequencing libraries.We have also developed a hybrid-selection protocol to further enrich the viral content of sequencing libraries.These protocols have enabled rapid deep sequencing of both Lassa and Ebola virus and are broadly applicable to other viral genomics studies.
We have developed a robust RNA sequencing method for generating complete de novo assemblies with intra-host variant calls of Lassa and Ebola virus genomes in clinical and biological samples. Our method uses targeted RNase H-based digestion to remove contaminating poly(rA) carrier and ribosomal RNA. This depletion step improves both the quality of data and quantity of informative reads in unbiased total RNA sequencing libraries. We have also developed a hybrid-selection protocol to further enrich the viral content of sequencing libraries. These protocols have enabled rapid deep sequencing of both Lassa and Ebola virus and are broadly applicable to other viral genomics studies.
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Mentions: First, we discovered that RNA samples extracted using commercial kits containing poly(rA) RNA carrier resulted in high-molecular-weight byproducts (Additional file 1: Figure S1A). To confirm that these byproducts came from carrier RNA, we added poly(rA) to RNA extracted without carrier and compared the resulting library to a poly(rA)-free control library from the same sample; the high-molecular-weight products were observed only when carrier RNA was added (Figure 1A). Poly(rA) also negatively impacted the raw Illumina sequencing data. As shown in Figure 1B, the median base quality dropped significantly about halfway through the forward and reverse 150-base reads, presumably due to poly(A) reads interfering with calibration of base-calling on the flow cell, while a poly(rA)-free library stayed well above a quality score of 25 until the end of the run.Figure 1