RAPIDprep: A Simple, Fast Protocol for RNA Metagenomic Sequencing of Clinical Samples
<p>RAPID<span class="html-italic">prep</span> development experiments. All results here are derived from the same sample extracts (RESP01-RESP03) run in duplicate and presented as mean values and error as standard deviation (SD). (<b>A</b>) The shaded bars are representative of the percentage of residual rRNA reads in the library following rRNA depletion with either an in-reaction cDNA synthesis method (grey) or a pre-cDNA hybridization approach (orange). The bars are clustered with respect to the sample they are derived from, labeled on the X-axis. (<b>B</b>) A comparison in total library yield, in nanomolar generated using Tapestation values, following a parallel experiment with a one-step and two-step second strand synthesis step using the Sequenase enzyme. The grey and orange shaded bars are representative of the one-step and two-step protocols, respectively. (<b>C</b>) Grey-shaded bars represent the total library yield of each sample under different library amplification cycling conditions. The X-axis is marked with the number of amplification cycles and is sub-grouped by source sample. (<b>D</b>) The duplication rate of reads generated in the final libraries following cycle titration; the number of cycles for each sample is indicated on the X-axis, and is sub-grouped by source sample.</p> "> Figure 2
<p>Filtered read distribution and classification across forty RAPID<span class="html-italic">prep</span> libraries. The sequence reads were classified into five categories: low-quality reads (blue), human rRNA reads (red), human non-rRNA (pink), non-human rRNA reads (green), and non-human non-rRNA reads (light green). Low-quality, human rRNA, human non-rRNA, and non-human rRNA were excluded from downstream analysis, and the non-human non-rRNA reads were the sole target reads for pathogen detection. Relative distribution was calculated by dividing the number of reads mapping to the relative category by the total number of reads for the individual library, before conversion into a percentage by multiplying the value by 100. The results were ordered by library number and grouped by sample type with a further key in grey shaded indicating the sample extraction platform used.</p> "> Figure 3
<p>Quantitative detection of SARS-COV-2 and RSV sequences. A simple linear-regression model was applied to both SARS-CoV-2 (<b>A</b>) and RSV (<b>B</b>) data sets with a line of best fit estimating the relationship between log-transformed reads per million (<sub>log</sub>RPM) and cycle threshold (CT) values. The linear-regression slope coefficient and the intercept parameter are printed on the top right of each plot, with R<sup>2</sup> calculated to measure the goodness of fit.</p> "> Figure 4
<p>Comparison of RAPID<span class="html-italic">prep</span> to commercial RNA library preparation kit. Using previously generated data for the kids SARI cohort, we compared the 24 most abundant species identified across both protocols for the same set of samples. An unclustered heatmap of microbial abundance (Z-score) is shown, with differences between samples identified by a deeper blue shading, while organisms conserved across samples are lighter blue through to red. A frequency histogram is overlayed on the color key and signifies the count of each Z score at any given point. Tick labels on the X-axis in the ICUXX format represent deep-RNA sequencing generated previously, while tick labels in the RAPIDXX format represent sequencing data generated in this study using the RAPID<span class="html-italic">prep</span> assay for the corresponding samples.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Specimens
2.2. RAPIDprep Assay
2.3. Development of Final Assay Conditions
2.4. Severe Acute Respiratory Infections in Children Cohort
2.5. Bioinformatic Analysis of RNA-mNGS Data
3. Results and Discussion
3.1. Development of the RAPIDprep Assay
3.1.1. rRNA Depletion
3.1.2. Double-Stranded cDNA Synthesis
3.1.3. Library Amplification
3.2. Application of the RAPIDprep Assay to a Panel of Respiratory Samples
3.3. Viral Sequence Identification, Genome Recovery, and Quantitative Performace
3.4. Comparison of RAPIDprep to Commercial Assay
3.5. Study Limitations
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Reagent | Catalogue | Supplier |
QIAseq FastSelect-rRNA HMR | 334385 | Qiagen, Hilden, Germany |
QIAseq FastSelect–5S/16S/23S | 335921 | |
Invitrogen SuperScript IV VILO Master Mix | 11756050 | Thermo Fisher, Waltham, MA, USA |
Sequenase Version 2.0 DNA Polymerase | 70775Y200UN | |
Invitrogen ezDNase Enzyme | 11766051 | |
Mag-Bind® TotalPure NGS | M1378-01 | Omega Biotek, Norcross, GA, USA |
Nextera XT DNA Library Preparation Kit | FC-131-1096 | Illumina, San Diego, CA, USA |
IDT® for Illumina DNA/RNA UD Indexes | 20027213 | |
iSeq 100 i1 Reagent v2 (300-cycle) | 20031371 |
In-Reaction | Pre-cDNA Hybridisation | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
rRNA | RESP01 | RESP02 | RESP03 | RESP01 | RESP02 | RESP03 | ||||||
Archaeal:16S | 3.5% | 2.9% | 7.5% | 7.3% | 6.1% | 4.0% | 0.0% | 0.0% | 0.1% | 0.1% | 0.2% | 0.1% |
Archaeal:23S | 10.9% | 9.5% | 19.2% | 19.9% | 22.0% | 16.2% | 0.1% | 0.1% | 0.8% | 0.7% | 1.6% | 1.4% |
Bacterial:5S | 0.7% | 0.8% | 0.2% | 0.2% | 0.4% | 0.6% | 1.0% | 1.3% | 0.9% | 0.8% | 1.4% | 1.3% |
Bacterial:16S | 0.7% | 0.6% | 2.1% | 2.1% | 3.0% | 2.2% | 0.1% | 0.0% | 0.2% | 0.2% | 0.2% | 0.1% |
Bacterial:23S | 3.5% | 3.1% | 10.1% | 10.7% | 20.7% | 18.1% | 0.2% | 0.3% | 2.2% | 2.1% | 4.0% | 3.9% |
Eukaryotic:5.8S | 0.5% | 0.5% | 1.1% | 1.1% | 0.9% | 0.8% | 0.0% | 0.0% | 0.1% | 0.1% | 0.1% | 0.1% |
Eukaryotic:18S | 14.0% | 11.9% | 22.0% | 22.1% | 11.2% | 7.7% | 0.3% | 0.4% | 0.9% | 0.8% | 0.6% | 0.5% |
Eukaryotic:28S | 8.5% | 6.7% | 12.6% | 13.0% | 6.6% | 4.4% | 0.2% | 0.2% | 0.7% | 0.7% | 0.4% | 0.4% |
rRNA levels | 0.0% | 5.0% | 10.0% | 15.0% | 20.0% | 25.0% |
Library. | Group | Virus | Type | Extraction Method | Library Yield (nM) | Data Output (Reads) |
---|---|---|---|---|---|---|
RAPID01 | COVID-19 | SARS-CoV-2 | Nasopharyngeal swab | Zymo Quick-RNA Viral | 8 | 16,810,302 |
RAPID02 | SARS-CoV-2 | Nasopharyngeal swab | Zymo Quick-RNA Viral | 34.7 | 11,620,222 | |
RAPID03 | SARS-CoV-2 | Nasopharyngeal swab | Zymo Quick-RNA Viral | 2.8 | 18,322,864 | |
RAPID04 | SARS-CoV-2 | Nasopharyngeal swab | Zymo Quick-RNA Viral | 2.4 | 12,707,642 | |
RAPID05 | SARS-CoV-2 | Nasopharyngeal swab | Zymo Quick-RNA Viral | 2.2 | 15,327,662 | |
RAPID06 | SARS-CoV-2 | Nasopharyngeal swab | Zymo Quick-RNA Viral | 13.2 | 15,271,010 | |
RAPID07 | SARS-CoV-2 | Nasopharyngeal swab | Zymo Quick-RNA Viral | 3.1 | 11,147,058 | |
RAPID08 | SARS-CoV-2 | Nasopharyngeal swab | Zymo Quick-RNA Viral | 6.1 | 9,453,054 | |
RAPID09 | SARS-CoV-2 | Nasopharyngeal swab | Zymo Quick-RNA Viral | 15.2 | 17,326,098 | |
RAPID10 | SARS-CoV-2 | Nasopharyngeal swab | Zymo Quick-RNA Viral | 9.1 | 15,531,486 | |
RAPID11 | SARS-CoV-2 | Nasopharyngeal swab | Zymo Quick-RNA Viral | 1.6 | 10,903,012 | |
RAPID12 | SARS-CoV-2 | Nasopharyngeal swab | Zymo Quick-RNA Viral | 16.2 | 12,670,186 | |
RAPID13 | Influenza A | pdmH1N1 | Viral culture | Zymo Quick-RNA Viral | 29.8 | 15,200,408 |
RAPID14 | pdmH1N1 | Viral culture | Zymo Quick-RNA Viral | 3.5 | 12,405,816 | |
RAPID15 | Mock community | None | Mixed culture | ZymoBIOMICS DNA/RNA Miniprep | 44.5 | 13,541,676 |
RAPID16 | None | Mixed culture | ZymoBIOMICS DNA/RNA Miniprep | 86 | 12,427,300 | |
RAPID17 | Kids SARI | Unknown | Nasopharyngeal aspirate | ZymoBIOMICS DNA/RNA Miniprep | 6.6 | 16,321,598 |
RAPID18 | Unknown | Nasopharyngeal aspirate | ZymoBIOMICS DNA/RNA Miniprep | 1.5 | 17,340,092 | |
RAPID19 | Unknown | Nasopharyngeal aspirate | ZymoBIOMICS DNA/RNA Miniprep | 1 | 15,464,422 | |
RAPID20 | Unknown | Nasopharyngeal aspirate | ZymoBIOMICS DNA/RNA Miniprep | 8.3 | 16,563,150 | |
RAPID21 | Unknown | Nasopharyngeal aspirate | ZymoBIOMICS DNA/RNA Miniprep | 1.6 | 24,661,800 | |
RAPID22 | Unknown | Nasopharyngeal aspirate | ZymoBIOMICS DNA/RNA Miniprep | 8.8 | 14,490,708 | |
RAPID23 | Unknown | Nasopharyngeal aspirate | ZymoBIOMICS DNA/RNA Miniprep | 3.3 | 28,187,178 | |
RAPID24 | Unknown | Nasopharyngeal aspirate | ZymoBIOMICS DNA/RNA Miniprep | 3.8 | 19,042,138 | |
RAPID25 | RSV | RSV | Nasopharyngeal swab | Roche MagNA Pure 96 Viral NA | 84.2 | 15,287,586 |
RAPID26 | RSV | Nasopharyngeal swab | Roche MagNA Pure 96 Viral NA | 94.1 | 19,473,790 | |
RAPID27 | RSV | Nasopharyngeal swab | Roche MagNA Pure 96 Viral NA | 97.5 | 16,302,456 | |
RAPID28 | RSV | Nasopharyngeal swab | Roche MagNA Pure 96 Viral NA | 80.1 | 14,524,914 | |
RAPID29 | RSV | Nasopharyngeal swab | Roche MagNA Pure 96 Viral NA | 67.9 | 17,923,728 | |
RAPID30 | RSV | Nasopharyngeal swab | Roche MagNA Pure 96 Viral NA | 49.3 | 13,466,538 | |
RAPID31 | RSV | Nasopharyngeal swab | Roche MagNA Pure 96 Viral NA | 56.7 | 12,192,164 | |
RAPID32 | RSV | Nasopharyngeal swab | Roche MagNA Pure 96 Viral NA | 61.8 | 17,199,224 | |
RAPID33 | Kids unknown | Unknown | Nasopharyngeal aspirate | ZymoBIOMICS DNA/RNA Miniprep | 22.2 | 14,839,480 |
RAPID34 | Unknown | Nasopharyngeal swab | Zymo Quick-RNA Viral | 31.4 | 14,806,470 | |
RAPID35 | Unknown | Nasopharyngeal swab | Zymo Quick-RNA Viral | 70.1 | 12,576,818 | |
RAPID36 | Unknown | Nasopharyngeal aspirate | Zymo Quick-RNA Viral | 10.9 | 13,418,996 | |
RAPID37 | Unknown | Vomitus | Zymo Quick-RNA Viral | 11.8 | 9,862,060 | |
RAPID38 | Unknown | Nasopharyngeal swab | Zymo Quick-RNA Viral | 48.1 | 11,384,408 | |
RAPID39 | Unknown | Nasopharyngeal swab | Zymo Quick-RNA Viral | 134.5 | 20,986,506 | |
RAPID40 | NTC | NTC | Water | N/A | 6.4 | 26,137,100 |
Library. | Virus | Type # | SARS-CoV2 | RSV-A | RSV-B | Flu-A pdmH1N1 | Flu-C | Rhinovirus | GB Virus C | CMV | HHV7 |
---|---|---|---|---|---|---|---|---|---|---|---|
RAPID01 | SARS-CoV-2 | NP swab | 5.80 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
RAPID02 | SARS-CoV-2 | NP swab | 3.49 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
RAPID03 | SARS-CoV-2 | NP swab | 3.50 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
RAPID04 | SARS-CoV-2 | NP swab | 5.89 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
RAPID05 | SARS-CoV-2 | NP swab | 3.98 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
RAPID06 | SARS-CoV-2 | NP swab | 5.92 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
RAPID07 | SARS-CoV-2 | NP swab | 5.82 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 1.15 | 0.00 | 0.00 |
RAPID08 | SARS-CoV-2 | NP swab | 5.93 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
RAPID09 | SARS-CoV-2 | NP swab | 5.98 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
RAPID10 | SARS-CoV-2 | NP swab | 4.90 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
RAPID11 | SARS-CoV-2 | NP swab | 4.81 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
RAPID12 | SARS-CoV-2 | NP swab | 5.98 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
RAPID13 | pdmH1N1 | Culture | 0.00 | 0.00 | 0.00 | 5.94 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
RAPID14 | pdmH1N1 | Culture | 0.00 | 0.00 | 0.00 | 5.83 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
RAPID15 | None | Culture | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
RAPID16 | None | Culture | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
RAPID17 | Unknown | NP aspirate | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.62 | 0.00 |
RAPID18 | Unknown | NP aspirate | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
RAPID19 | Unknown | NP aspirate | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
RAPID20 | Unknown | NP aspirate | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
RAPID21 | Unknown | NP aspirate | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
RAPID22 | Unknown | NP aspirate | 0.00 | 0.00 | 0.00 | 0.00 | 5.77 | 0.00 | 0.00 | 0.00 | 0.00 |
RAPID23 | Unknown | NP aspirate | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
RAPID24 | Unknown | NP aspirate | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
RAPID25 | RSV | NP swab | 0.00 | 0.00 | 2.64 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
RAPID26 | RSV | NP swab | 0.00 | 3.96 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
RAPID27 | RSV | NP swab | 0.00 | 0.00 | 3.25 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
RAPID28 | RSV | NP swab | 0.00 | 2.89 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
RAPID29 | RSV | NP swab | 0.00 | 0.00 | 4.09 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
RAPID30 | RSV | NP swab | 0.00 | 5.25 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
RAPID31 | RSV | NP swab | 0.00 | 4.67 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
RAPID32 | RSV | NP swab | 0.00 | 0.00 | 3.14 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
RAPID33 | Unknown | NP aspirate | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 5.61 | 0.00 | 0.00 | 0.00 |
RAPID34 | Unknown | NP swab | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 5.94 | 0.00 | 0.00 | 0.00 |
RAPID35 | Unknown | NP swab | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 6.00 | 0.00 | 0.00 | 0.00 |
RAPID36 | Unknown | NP aspirate | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 2.35 |
RAPID37 | Unknown | Vomitus | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
RAPID38 | Unknown | NP swab | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 4.79 | 0.00 | 0.00 | 0.00 |
RAPID39 | Unknown | NP swab | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 5.98 | 0.00 | 0.00 | 0.00 |
RAPID40 | NTC | Water | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
Log-RPM | 6.00 | 5.00 | 4.00 | 3.00 | 2.00 | 1.00 | 0.00 |
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Tulloch, R.L.; Kim, K.; Sikazwe, C.; Michie, A.; Burrell, R.; Holmes, E.C.; Dwyer, D.E.; Britton, P.N.; Kok, J.; Eden, J.-S. RAPIDprep: A Simple, Fast Protocol for RNA Metagenomic Sequencing of Clinical Samples. Viruses 2023, 15, 1006. https://doi.org/10.3390/v15041006
Tulloch RL, Kim K, Sikazwe C, Michie A, Burrell R, Holmes EC, Dwyer DE, Britton PN, Kok J, Eden J-S. RAPIDprep: A Simple, Fast Protocol for RNA Metagenomic Sequencing of Clinical Samples. Viruses. 2023; 15(4):1006. https://doi.org/10.3390/v15041006
Chicago/Turabian StyleTulloch, Rachel L., Karan Kim, Chisha Sikazwe, Alice Michie, Rebecca Burrell, Edward C. Holmes, Dominic E. Dwyer, Philip N. Britton, Jen Kok, and John-Sebastian Eden. 2023. "RAPIDprep: A Simple, Fast Protocol for RNA Metagenomic Sequencing of Clinical Samples" Viruses 15, no. 4: 1006. https://doi.org/10.3390/v15041006