Targeted NGS-Based Analysis of Pneumocystis jirovecii Reveals Novel Genotypes
Abstract
:1. Introduction
2. Materials and Methods
2.1. Human Samples
2.2. DNA Extraction
2.3. Library Preparation
2.4. DNA Sequencing
2.5. Sequencing Analysis
3. Results
3.1. Library Prep Validation
3.2. Meu 10 Genotype
3.3. FourF5 Genotype
3.4. Mitochondrial Genotypes
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Sequence Name | Target: Gene Name/Murina Ortholog | Target Length | Sequence 5′ to 3′ | Primer Notes |
---|---|---|---|---|
RHH.W4300B6A5BDF041 | N/A | N/A | N/A | Custom rhAmpSeq™ FWD Panel |
RH.CF78E0180F584D9Z0Z.F | T551_03199/Arp9 | 134 | /rhSeq-f/TCA TAG CCC ACA TGA ATA ACA ATT rCCA GA/GT4/ | Forward |
RH.A2C31F6013C24B7Z0Z.F | T551_03199/Arp9 | 148–152 | /rhSeq-f/GTT CAA TGC TGA CAC TGT TTA TCrG CCA T/GT4/ | Forward |
RH.1B63D59F4F974A1Z0Z.F | T551_03038/meiotically up-regulated gene 117 protein | 138–142 | /rhSeq-f/GGT TTC TGA GAA AGA TTT AGC CAT ATrG TAA C/GT1/ | Forward |
RH.FD38EF6B8AEA421Z0Z.F | T551_00303/GTP binding | 148–150 | /rhSeq-f/GAT CCA CTG TCC AGA CAA TTrG CAT C/GT1/ | Forward |
RH.103374AC8028435Z0Z.F | T551_02533/Meu10 | 148 | /rhSeq-f/ATA GGA GTG GTT TCC GTC TTT rCTT TC/GT3/ | Forward |
RH.E0CE477C4B19464Z0Z.F | T551_00896/Four F5 protein | 150–153 | /rhSeq-f/GAG AAA ATG ATA GAC TTA GGG CTrC AAA A/GT4/ | Forward |
RH.316DF1391E794B9Z0Z.F | T551_01756/Acrl | 148–149 | /rhSeq-f/CAG AAG ATG AAT TGC CAC CAA rACA TT/GT4/ | Forward |
RH.E45C51A8E1AB462Z0Z.F | T551_01755/Serine protease | 110–112 | /rhSeq-f/CAT CTG CAG GTA AAG ACA AGA AArC CAG A/GT1/ | Forward |
RH.AD8B9A3AFA344A3Z0Z.F | T551_01830/heat shock protein 70 | 142–144 | /rhSeq-f/CCC GAA TTT CCG TCC AAT rCAG AC/GT2/ | Forward |
RH.47196AD4997D471Z0Z.F | T551_02650/SUMO-targeted ubiquitin-protein ligase E3 Slx8 | 136–138 | /rhSeq-f/TGA CAC ACA AAG GCC ATT TAT TrCT GTC/GT1/ | Forward |
RH.AEC03A6FD9A64E0Z0Z.F | T551_03158/DNAJ | 135–136 | /rhSeq-f/CCG AAT TGG AAC TCA CTT TAC TrCG AAG/GT3/ | Forward |
RH.E8A3CF8496644ACZ0Z.F | T551_02309/GSC-1 | 129–134 | /rhSeq-f/TCT GGA GTA AAT CGA ACT TGA TTT rGCT TC/GT4/ | Forward |
RH.8DF9507B0841432Z0Z.F | mito NAD6 | 149–153 | /rhSeq-f/CAA TTC TGG TTG TAT CTT CAA GAA ATC rCTG TA/GT2/ | Forward |
RH.3E370038B0CB4B3Z0Z.F | mito NAD1 | 151–156 | /rhSeq-f/GCT TTA TTA GGT TCC TTG TGA AGT ArCT GCT/GT3/ | Forward |
RH.DDFCD6AD33494FBZ0Z.F | cytochrome c oxidase subunit 1/ atp8 | 155–160 | /rhSeq-f/TGA TTT CAC CAC CAG CTT TCrC ATG C/GT1/ | Forward |
RH.FF72BC52B1E14BCZ0Z.F | atp6 | 155–160 | /rhSeq-f/CTT CAC ATC TGG TCT TTA CTG TCrG CTT T/GT3/ | Forward |
RH.E67274DA8AD04D9Z0Z.F | cytochrome c oxidase subunit 3 | 151–156 | /rhSeq-f/CTA GCT TTT ACT CTT TTA CAG GGT rGTG GA/GT4/ | Forward |
RH.EF9CF32F289649AZ0Z.F | atp9 | 149–152 | /rhSeq-f/GGT TCA GGG TTA GCT ACA ATT rGGA TT/GT4/ | Forward |
RH.99229D6013E14CBZ0Z.F | NADH dehydrogenase subunit 4L/nad5 | 151–152 | /rhSeq-f/TCC ATG GCT TTA GAT GAT TTA GAA GrGA CAG/GT1/ | Forward |
RH.795964E1E42344EZ0Z.F | Nad4 | 125–128 | /rhSeq-f/TCA CTC CTT TGG TCT ATA CTG TrCT GTG/GT1/ | Forward |
RHH.W4300B6A5BDF041 | N/A | N/A | N/A | Custom rhAmpSeq™ REV Panel |
RH.CF78E0180F584D9Z0Z.R | T551_03199/Arp9 | 134 | /rhSeq-r/CGT GAT AAA GAA CTT GCA ACA TGrC ACA T/GT1/ | Reverse |
RH.A2C31F6013C24B7Z0Z.R | T551_03199/Arp9 | 148–152 | /rhSeq-r/GAG AGT ATT TGG CTC AAA AAG AAG rCGG AG/GT4/ | Reverse |
RH.1B63D59F4F974A1Z0Z.R | T551_03038/meiotically up-regulated gene 117 protein | 138–142 | /rhSeq-r/TCT ACT CTA TTA ATG TAT TCT GAC CCA rCAA GG/GT4/ | Reverse |
RH.FD38EF6B8AEA421Z0Z.R | T551_00303/GTP binding | 148–150 | /rhSeq-r/GGT TAT GAC AAT TTC GCC GArC CGT T/GT2/ | Reverse |
RH.103374AC8028435Z0Z.R | T551_02533/Meu10 | 148 | /rhSeq-r/TGA CCA AGT CGC TGT TAT TGT rATA TG/GT2/ | Reverse |
RH.E0CE477C4B19464Z0Z.R | T551_00896/Four F5 protein | 150–153 | /rhSeq-r/GAT GTT TGC TTG GCT AAG GTA rCAG AT/GT1/ | Reverse |
RH.316DF1391E794B9Z0Z.R | T551_01756/Acrl | 148–149 | /rhSeq-r/TCC CAT CGT AAA AGG CGT AAT rCTT GA/GT1/ | Reverse |
RH.E45C51A8E1AB462Z0Z.R | T551_01755/Serine protease | 110–112 | /rhSeq-r/TGA ACA AAT GAA TGC GAC AAT AGT rCCC AA/GT2/ | Reverse |
RH.AD8B9A3AFA344A3Z0Z.R | T551_01830/heat shock protein 70 | 142–144 | /rhSeq-r/TCA AGG GAA TCG AAC CAC ArCC ATC/GT4/ | Reverse |
RH.47196AD4997D471Z0Z.R | T551_02650/SUMO-targeted ubiquitin-protein ligase E3 Slx8 | 136–138 | /rhSeq-r/TTT GTT TGG TGC AAG CAT TAT TTrC AAG G/GT2/ | Reverse |
RH.AEC03A6FD9A64E0Z0Z.R | T551_03158/DNAJ | 135–136 | /rhSeq-r/CGT GAC CAG GAT ACG ATA TCT rCAT GG/GT3/ | Reverse |
RH.E8A3CF8496644ACZ0Z.R | T551_02309/GSC-1 | 129–134 | /rhSeq-r/CCT TTA GAA TCT GCA ATA TAT CGT TrGG AAG/GT4/ | Reverse |
RH.8DF9507B0841432Z0Z.R | mito NAD6 | 149–153 | /rhSeq-r/GCT ATA GCT CCA ACA TAT ACA GTA ATrA TAT G/GT4/ | Reverse |
RH.3E370038B0CB4B3Z0Z.R | mito NAD1 | 151–156 | /rhSeq-r/GGC AAA ACA AAC CAA ATA GCT TTT TrGT TCT/GT1/ | Reverse |
RH.DDFCD6AD33494FBZ0Z.R | cytochrome c oxidase subunit 1/ atp8 | 155–160 | /rhSeq-r/TGT CTA GGC AGA ACC CAT TTA rGCG AA/GT2/ | Reverse |
RH.FF72BC52B1E14BCZ0Z.R | atp6 | 155–160 | /rhSeq-r/ACA AAG AAG GAG AAG AAT TTC AGA rCCA TG/GT1/ | Reverse |
RH.E67274DA8AD04D9Z0Z.R | cytochrome c oxidase subunit 3 | 151–156 | /rhSeq-r/AGT CCT ACA GAT AAA AAG ATA GCA rCCC AC/GT1/ | Reverse |
RH.EF9CF32F289649AZ0Z.R | atp9 | 149–152 | /rhSeq-r/GCT TCT GCT AGA GCA AAT CCrC AAG A/GT1/ | Reverse |
RH.99229D6013E14CBZ0Z.R | NADH dehydrogenase subunit 4L/nad5 | 151–152 | /rhSeq-r/ACA CCG ATT TTA TGT CCT AAC AAA rCCT AC/GT4/ | Reverse |
RH.795964E1E42344EZ0Z.R | Nad4 | 125–128 | /rhSeq-r/GCC AAA GCC ATA TGT CCA ATA rGAA GA/GT4/ | Reverse |
i5_IDT-1 | N/A | N/A | AAT GAT ACG GCG ACC ACC GAG ATC TAC ACA TAT GCG CAC ACT CTT TCC CTA CAC GAC | rhAmpSeq™ Index 1 (i5) Primer |
i5_IDT-2 | N/A | N/A | AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT GGT ACA GAC ACT CTT TCC CTA CAC GAC | rhAmpSeq™ Index 2 (i5) Primer |
i7_IDT-1 | N/A | N/A | CAA GCA GAA GAC GGC ATA CGA GAT ACG ATC AGG TGA CTG GAG TTC AGA CGT GT | rhAmpSeq™ Index 1 (i7) Primer |
i7_IDT-2 | N/A | N/A | CAA GCA GAA GAC GGC ATA CGA GAT TCG AGA GTG TGA CTG GAG TTC AGA CGT GT | rhAmpSeq™ Index 2 (i7) Primer |
i7_IDT-3 | N/A | N/A | CAA GCA GAA GAC GGC ATA CGA GAT CTA GCT CAG TGA CTG GAG TTC AGA CGT GT | rhAmpSeq™ Index 3 (i7) Primer |
i7_IDT-4 | N/A | N/A | CAA GCA GAA GAC GGC ATA CGA GAT ATC GTC TCG TGA CTG GAG TTC AGA CGT GT | rhAmpSeq™ Index 4 (i7) Primer |
i7_IDT-5 | N/A | N/A | CAA GCA GAA GAC GGC ATA CGA GAT TCG ACA AGG TGA CTG GAG TTC AGA CGT GT | rhAmpSeq™ Index 5 (i7) Primer |
i7_IDT-6 | N/A | N/A | CAA GCA GAA GAC GGC ATA CGA GAT CCT TGG AAG TGA CTG GAG TTC AGA CGT GT | rhAmpSeq™ Index 6 (i7) Primer |
i7_IDT-7 | N/A | N/A | CAA GCA GAA GAC GGC ATA CGA GAT ATC ATG CGG TGA CTG GAG TTC AGA CGT GT | rhAmpSeq™ Index 7 (i7) Primer |
i7_IDT-8 | N/A | N/A | CAA GCA GAA GAC GGC ATA CGA GAT TGT TCC GTG TGA CTG GAG TTC AGA CGT GT | rhAmpSeq™ Index 8 (i7) Primer |
i7_IDT-9 | N/A | N/A | CAA GCA GAA GAC GGC ATA CGA GAT ATT AGC CGG TGA CTG GAG TTC AGA CGT GT | rhAmpSeq™ Index 9 (i7) Primer |
i7_IDT-10 | N/A | N/A | CAA GCA GAA GAC GGC ATA CGA GAT CGA TCG ATG TGA CTG GAG TTC AGA CGT GT | rhAmpSeq™ Index 10 (i7) Primer |
i7_IDT-11 | N/A | N/A | CAA GCA GAA GAC GGC ATA CGA GAT GAT CTT GCG TGA CTG GAG TTC AGA CGT GT | rhAmpSeq™ Index 11 (i7) Primer |
i7_IDT-12 | N/A | N/A | CAA GCA GAA GAC GGC ATA CGA GAT AGG ATA GCG TGA CTG GAG TTC AGA CGT GT | rhAmpSeq™ Index 12 (i7) Primer |
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Pungan, D.; Eddens, T.; Song, K.; Lakey, M.A.; Crovetto, N.S.; Arora, S.K.; Husain, S.; Kolls, J.K. Targeted NGS-Based Analysis of Pneumocystis jirovecii Reveals Novel Genotypes. J. Fungi 2022, 8, 863. https://doi.org/10.3390/jof8080863
Pungan D, Eddens T, Song K, Lakey MA, Crovetto NS, Arora SK, Husain S, Kolls JK. Targeted NGS-Based Analysis of Pneumocystis jirovecii Reveals Novel Genotypes. Journal of Fungi. 2022; 8(8):863. https://doi.org/10.3390/jof8080863
Chicago/Turabian StylePungan, Dora, Taylor Eddens, Kejing Song, Meredith A. Lakey, Nicolle S. Crovetto, Simran K. Arora, Shahid Husain, and Jay K. Kolls. 2022. "Targeted NGS-Based Analysis of Pneumocystis jirovecii Reveals Novel Genotypes" Journal of Fungi 8, no. 8: 863. https://doi.org/10.3390/jof8080863