Utilizing PacBio Iso-Seq for Novel Transcript and Gene Discovery of Abiotic Stress Responses in Oryza sativa L.
Abstract
:1. Introduction
2. Results
2.1. De Novo Reconstruction of Transcriptomes
2.2. Collapsing Redundant Isoforms
2.3. Evaluation of Reconstructed Transcriptomes
2.4. Functional Annotation
2.5. Common and Specific Transcripts among Cultivars
2.6. Differential Gene Expression Analysis for Aus Specific Transcripts
3. Discussion
3.1. Sequencing Performance
3.2. Collapsing Redundant Transcripts and Transcriptome Quality Assessment
3.3. Common Transcripts and Differential Gene Expression Analysis
4. Materials and Methods
4.1. Plant Material
4.2. RNA Extraction and Sequencing
4.3. De novo Transcriptome Reconstruction
- ccs $in.subreads.bam $out.bam --noPolish --minPasses=1
- lima $in.xml primer.fasta $out.demux.ccs.bam --isoseq --no-pbi --dump-clips
- isoseq3 cluster $in.demux.ccs.bam $out.unpolished.bam
- isoseq3 polish $in.unpolished.bam $out.polished.bam
4.4. Genome References
4.5. InDel Analysis
4.6. Collapsing Redundant Isoforms
4.7. BUSCO Analysis
4.8. Phylogenetic Analysis
4.9. Comparison of Reconstructed Transcriptomes
4.10. Functional Annotation
4.11. Determination of Common Overlap
4.12. Differential Gene Expression Analysis
4.13. Graphical Visualization
4.14. Availability of Data and Material
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
bp | Basepairs |
BUSCO | Benchmarking Universal Single-Copy Orthologs |
FLNC | Full-Length Non-Chimeric |
GB | Gigabases |
HNT | High Night Temperature |
HQ | High Quality |
InDel | Insertion/Deletion |
IRGSP | International Rice Genome Sequencing Project |
IsoSeq | Isoform Sequencing |
LQ | Low Quality |
ORF | Open Reading Frame |
RNA-Seq | RNA Sequencing |
SMRT | Single-Molecule, Real-Time |
SNP | Single Nucleotide Polymorphism |
Appendix A
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Cultivar | ssp. | ID No. | Organ | Set-up | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
FL | LE | PA | FS | DS | SH | RO | SO | PP | AN | CC | F | NH | |||
Dular | aus | IRGC 636 | X | X | X | X | |||||||||
N22 | IRTP 3911 | X | X | X | X | ||||||||||
Anjali | indica | IRTP 23206 | X | X | X | X | |||||||||
IR62266-42-6-2 | IRGC 117597 | X | X | X | X | X | X | X | |||||||
IR64 | IRTP 12158 | X | X | X | X | X | X | X | X | ||||||
IR72 | IRTP 14747 | X | X | X | X | X | X | X | |||||||
CT9993-5-10-1M | japonica | IRIS 71-1229921 | X | X | X | X | X | X | X | ||||||
M202 | IRGC 77142 | X | X | X | X | X | X | X | |||||||
Moroberekan | IRGC 12048 | X | X | X | X | X | X | X | X | ||||||
Nipponbare | IRGC 12731 | X | X | X | X | X |
Cultivar | ssp. | PB | GB | FLNC | HQ | LQ | HQ after Filtering |
---|---|---|---|---|---|---|---|
Dular | aus | 2 | 18.46 | 460,340 | 42,252 | 1960 | 41,396 |
N22 | 3 | 24.17 | 736,747 | 54,572 | 1807 | 52,333 | |
Anjali | indica | 2 | 15.49 | 481,094 | 40,208 | 1732 | 39,438 |
IR62266-42-6-2 | 2 | 22.48 | 649,085 | 50,569 | 1659 | 50,510 | |
IR64 | 2 | 21.97 | 622,881 | 49,633 | 1279 | 49,327 | |
IR72 | 2 | 20.31 | 554,872 | 44,176 | 2170 | 44,049 | |
CT9993-5-10-1M | japonica | 2 | 20.81 | 620,595 | 48,537 | 1465 | 48,401 |
M202 | 2 | 24.07 | 656,740 | 48,836 | 1501 | 48,676 | |
Moroberekan | 2 | 24.51 | 675,251 | 54,684 | 1721 | 54,594 | |
Nipponbare | 3 | 15.65 | 544,792 | 37,951 | 1233 | 37,535 |
Cultivar | ssp. | Reference | # Tr. | Reference-Based | Reference-Free | |||
---|---|---|---|---|---|---|---|---|
TAMA | cDNA Cupcake | Unmapped | Cogent | Unmapped | ||||
Dular | aus | n22 | 41,396 | 13,995 | 18,239 | 313 | 13,107 | 7340 |
N22 | 52,333 | 18,787 | 23,954 | 149 | 19,026 | 6603 | ||
Anjali | indica | S498 | 39,438 | 14,371 | 18,170 | 178 | 13,237 | 6476 |
IR62266-42-6-2 | 50,510 | 18,926 | 23,803 | 220 | 18,773 | 6913 | ||
IR64 | 49,327 | 19,064 | 23,435 | 1911 | 17,874 | 7979 | ||
IR72 | 44,049 | 15,954 | 20,646 | 143 | 15,251 | 7426 | ||
CT9993-5-10-1M | japonica | Nipponbare | 48,401 | 18,789 | 23,415 | 223 | 18,359 | 6611 |
M202 | 48,676 | 18,925 | 23,670 | 240 | 18,091 | 6695 | ||
Moroberekan | 54,594 | 20,604 | 26,009 | 268 | 20,378 | 7358 | ||
Nipponbare | 37,535 | 16,584 | 19,674 | 42 | 14,345 | 5441 |
Cultivar | ssp. | # GL | # TR | # TR/GL | Total # bp | Min [bp] | Max [bp] | Median [bp] | GC [%] |
---|---|---|---|---|---|---|---|---|---|
Dular | aus | 10,511 | 14,255 | 1.4 | 15,447,641 | 56 | 4551 | 986 | 50.87 |
N22 | 13,343 | 18,913 | 1.4 | 26,290,969 | 62 | 5911 | 1295 | 52.26 | |
Anjali | indica | 10,616 | 14,499 | 1.4 | 17,717,403 | 75 | 4216 | 1156 | 51.99 |
IR62266-42-6-2 | 13,227 | 19,093 | 1.4 | 26,791,848 | 51 | 7190 | 1314 | 51.37 | |
IR64 | 15,011 | 20,672 | 1.4 | 28,663,408 | 56 | 6919 | 1299 | 52.76 | |
IR72 | 11,647 | 16,081 | 1.4 | 19,678,018 | 53 | 5475 | 1149 | 51.16 | |
CT9993-5-10-1M | japonica | 13,354 | 18,963 | 1.4 | 26,757,988 | 55 | 5752 | 1318 | 51.97 |
M202 | 13,143 | 19,105 | 1.5 | 26,258,012 | 59 | 6644 | 1287 | 51.74 | |
Moroberekan | 14,324 | 20,803 | 1.5 | 28,446,682 | 57 | 7072 | 1278 | 51.80 | |
Nipponbare | 11,366 | 16,622 | 1.5 | 24,760,098 | 75 | 6035 | 1394 | 52.60 | |
IRGSP | japonica | 38,866 | 45,660 | 1.2 | 69,184,066 | 30 | 16,029 | 1385 | 51.24 |
Cultivar | ssp. | Mercator | Blastx | Blastp | PFAM | GO | No Annotation | Homologs WS |
---|---|---|---|---|---|---|---|---|
Dular | aus | 61.60 | 65.17 | 59.57 | 59.81 | 37.98 | 27.60 | 90.54 |
N22 | 68.40 | 72.05 | 68.43 | 70.01 | 45.52 | 19.24 | 91.33 | |
Anjali | indica | 65.77 | 69.46 | 65.43 | 66.90 | 43.06 | 22.03 | 89.82 |
IR62266-46-6-2 | 68.08 | 71.53 | 67.16 | 68.64 | 44.85 | 20.19 | 91.19 | |
IR64 | 67.78 | 71.27 | 67.37 | 69.55 | 45.31 | 20.23 | 82.03 | |
IR72 | 63.55 | 67.20 | 62.26 | 63.78 | 41.22 | 24.96 | 88.54 | |
CT9993-5-10-1M | japonica | 68.57 | 71.80 | 67.58 | 69.24 | 45.01 | 19.62 | 92.43 |
M202 | 67.78 | 71.08 | 66.69 | 67.97 | 44.44 | 20.68 | 90.71 | |
Moroberekan | 65.72 | 69.03 | 64.66 | 66.85 | 43.42 | 22.37 | 91.68 | |
Nipponbare | 71.25 | 74.35 | 70.26 | 72.16 | 47.59 | 16.81 | 91.31 |
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Schaarschmidt, S.; Fischer, A.; Lawas, L.M.F.; Alam, R.; Septiningsih, E.M.; Bailey-Serres, J.; Jagadish, S.V.K.; Huettel, B.; Hincha, D.K.; Zuther, E. Utilizing PacBio Iso-Seq for Novel Transcript and Gene Discovery of Abiotic Stress Responses in Oryza sativa L. Int. J. Mol. Sci. 2020, 21, 8148. https://doi.org/10.3390/ijms21218148
Schaarschmidt S, Fischer A, Lawas LMF, Alam R, Septiningsih EM, Bailey-Serres J, Jagadish SVK, Huettel B, Hincha DK, Zuther E. Utilizing PacBio Iso-Seq for Novel Transcript and Gene Discovery of Abiotic Stress Responses in Oryza sativa L. International Journal of Molecular Sciences. 2020; 21(21):8148. https://doi.org/10.3390/ijms21218148
Chicago/Turabian StyleSchaarschmidt, Stephanie, Axel Fischer, Lovely Mae F. Lawas, Rejbana Alam, Endang M. Septiningsih, Julia Bailey-Serres, S. V. Krishna Jagadish, Bruno Huettel, Dirk K. Hincha, and Ellen Zuther. 2020. "Utilizing PacBio Iso-Seq for Novel Transcript and Gene Discovery of Abiotic Stress Responses in Oryza sativa L." International Journal of Molecular Sciences 21, no. 21: 8148. https://doi.org/10.3390/ijms21218148