Long-Read High-Throughput Sequencing (HTS) Revealed That the Sf-Rhabdovirus X+ Genome Contains a 3.7 kb Internal Duplication
Abstract
:1. Introduction
2. Materials and Methods
2.1. Sf9 Cells and Sf9 Cell Clones
2.2. RT-PCR Analysis
2.3. PacBio HTS Sequencing
2.4. Oxford Nanopore HTS Sequencing
2.5. Illumina HiSeq HTS Sequencing
3. Results
3.1. Identification of X+3.7 in Sf9 Cell Transcriptome
3.2. Identification of X+3.7 Produced by Sf9 X+ Cell Clones
3.3. Oxford Nanopore Sequencing of the X+3.7 Virus Produced from Sf-2-20 Cell Clone
3.4. Characterization of X+3.7 Genome Structure
3.5. Analysis of HTS Long-Reads and Short-Reads for Assembly of Sf- Rhabdovirus X+3.7 Genome
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Sample IDs | Total No. of Reads | No. of Reads Past Q7 Filter | Average Q7 Past Read Length (bp) | No. of Rhabdovirus Reads (CLC) | Average Rhabdovirus Read Length (bp) |
---|---|---|---|---|---|
2B | 28,062 | 5679 | 831 | 562 | 2481 |
2A | 233,281 | 93,287 | 570 | 92 | 2744 |
1 | 571,301 | 193,530 | 509 | 229 | 2025 |
Combined | 292,496 | 883 |
Read Length (bp) | Aligned Region in KF947078 3→5′ | Identity (%) 1 | Gaps (%) 1 |
---|---|---|---|
16,689 | 659–8523^GA^4767–13,526 (338) 2 | 92.1 | 5.5 |
12,529-R 3 | (83)-8523^GA^4767–8673 | 93.0 | 4.9 |
11,631 | 353–8523^GA^4767–8295 | 93.5 | 4.6 |
11,247-R | (84)-8523^GA^4767–7555 | 91.9 | 5.6 |
9135 | (65)-8523^GA^4767–5650 | 88.2 | 8.0 |
4634-R | 4407–8523^GA^4767–5375 | 91.1 | 6.5 |
4537 | 7054–8523^GA^4767–7613 | 91.4 | 6.2 |
2361 | 6297–8523^GA^4767–4895 | 88.6 | 7.2 |
916-R | 8096–8523^GA^4767–5275 | 92.8 | 5.7 |
Putative Genes | Location in KF947078 (nt) | Location in X+3.7 (nt) |
---|---|---|
N | 81–1646 | 90–1718 |
P | 1726–2871 | 1798–2943 |
M | 3088–4014 | 3160–4086 |
G | 4140–5972 | 4212–6044 |
X | 6161–6490 | 6233–6562 |
L1 | 6833–7074 | 6905–7153 |
L2 | 7211–7740 | 7290–7817 |
L3/G1 | 8505–8523^GA^4767–5972 | 8582–9808 |
X1 | 6161–6490 | 9997–10,326 |
L | 6833–13,255 | 10,669–17,091 |
Reference X+ Genome | X+3.7 Genome | ||||
---|---|---|---|---|---|
Primary Sequence | Duplication Sequence | ||||
Location (ORF) | nt | Location (ORF) | nt | Location (ORF) | nt |
4878 (G) | T | 4950 (G) | T | 8714 (L3/G1) | C |
4905 (G) | C | 4977 (G) | C | 8741 (L3/G1) | T |
4908 (G) | G | 4980 (G) | G | 8744 (L3/G1) | A |
5003 (G) | G | 5075 (G) | G | 8839 (L3/G1) | A |
5036 (G) | G | 5108 (G) | G | 8872 (L3/G1) | A |
5578 (G) | G | 5650 (G) | G | 9414 (L3/G1) | A |
5687 (G) | G | 5759 (G) | G | 9523 (L3/G1) | A |
5817 (G) | A | 5889 (G) | A | 9653 (L3/G1) | G |
6333 (X) | C | 6405 (X) | C | 10,169 (X1) | A |
6354 (X) | A | 6426 (X) | A | 10,190 (X1) | T |
6732 (X-L) | C | 6804 (X-L) | T | 10,568 (X1-L) | C |
7016^7017 (L) | - | 7089–7094 (L1) | AAAACC | 10,852^10,853 (L) | - |
7018 (L) | A | 7096 (L1) | C | 10,854 (L) | A |
7050^7051 (L) | - | 7129 (L1) | G | 10,886^10,887 (L) | - |
7452 (L) | C | 7531 (L2) | T | 11,288 (L) | C |
7624 (L) | T | 7703 (L2) | G | 11,460 (L) | T |
7699–7700 (L) | AG | 7777^7778 (L2) | -- | 11,535–11,536 (L) | AG |
8052 (L) | C | 8129 (L2-L3/G1) | T | 11,888 (L) | C |
8070 (L) | G | 8147 (L2-L3/G1) | A | 11,906 (L) | G |
8104 (L) | G | 8181 (L2-L3/G1) | T | 11,940 (L) | G |
8164 (L) | C | 8241 (L2-L3/G1) | T | 12,000 (L) | C |
8189 (L) | G | 8266 (L2-L3/G1) | A | 12,025 (L) | G |
8351 (L) | G | 8428 (L2-L3/G1) | A | 12,187 (L) | G |
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Ma, H.; Bosma, T.J.; Khan, A.S. Long-Read High-Throughput Sequencing (HTS) Revealed That the Sf-Rhabdovirus X+ Genome Contains a 3.7 kb Internal Duplication. Viruses 2023, 15, 1998. https://doi.org/10.3390/v15101998
Ma H, Bosma TJ, Khan AS. Long-Read High-Throughput Sequencing (HTS) Revealed That the Sf-Rhabdovirus X+ Genome Contains a 3.7 kb Internal Duplication. Viruses. 2023; 15(10):1998. https://doi.org/10.3390/v15101998
Chicago/Turabian StyleMa, Hailun, Trent J. Bosma, and Arifa S. Khan. 2023. "Long-Read High-Throughput Sequencing (HTS) Revealed That the Sf-Rhabdovirus X+ Genome Contains a 3.7 kb Internal Duplication" Viruses 15, no. 10: 1998. https://doi.org/10.3390/v15101998