188:. During the construction of the PET library, the fragments can be selected to all be of a certain size. After mapping, the PET sequences are thus expected to be consistently a particular distance away from each other. A discrepancy from this distance indicates a structural variation between the PET sequences. For example (Figure on the right): a deletion in the sequenced genome will have reads that map further away than expected in the reference genome as the reference genome will have a segment of DNA that is not present in the sequenced genome.
137:
168:. Anchoring one half of the pair uniquely to a single location in the genome allows mapping of the other half that is ambiguous. Ambiguous reads are those that map to more than a single location. This increased efficiency reduces the cost of sequencing as these ambiguous sequences, or reads, would normally be discarded. The connectivity of PET sequences also allows detection of structural variations:
145:
70:
251:: transcripts, gene structures, and gene expressions. The PET library is generated using full length cDNAs, so the ditags represent the 5’ capped and the 3’ polyA tail signatures of individual transcripts. Therefore, RNA-PET is especially useful for demarcating the boundaries of transcription units. This will help identify alternative transcription start sites and
52:(discussed below) used to produce PETs give longer tags (18/20 base pairs and 25/27 base pairs) but sequences of 50–100 base pairs would be optimal for both mapping and cost efficiency. After extracting the PETs from many DNA fragments, they are linked (concatenated) together for efficient sequencing. On average, 20–30 tags could be sequenced with the
279:
sites. The advantages of PET sequencing over these methods are that PET identify both ends of the transcripts and, at the same time, provide more specificity when mapping back to the genome. Sequencing the cDNAs can reveal the structures of transcripts in great details, but this approach is much more
60:
that has short read lengths and higher throughput. The main advantages of PET sequencing are its reduced cost by sequencing only short fragments, detection of structural variants in the genome, and increased specificity when aligning back to the genome compared to single tags, which involves only one
110:
Instead of cloning, adaptors containing the endonuclease sequence are ligated to the ends of fragmented genomic DNA or cDNA. The molecules are then self-circularized and digested with endonuclease, releasing the PET. Before sequencing, these PETs are ligated to adaptors to which PCR primers anneal
127:
cut downstream of their target binding sites. MmeI cuts 18/20 base pairs downstream and EcoP15I cuts 25/27 base pairs downstream. As these restriction enzymes bind at their target sequences located in the adaptors, they cut and release vectors that contain short sequences of the fragment or cDNA
97:
making the PET library. PET sequences are obtained by purifying plasmid and digesting with specific endonuclease leaving two short sequences on the ends of the vectors. Under intramolecular (dilute) conditions, vectors are re-circularized and ligated, leaving only the ditags in the vector. The
111:
for amplification. The advantage of cloning based construction of the library is that it maintains the fragments or cDNA intact for future use. However, the construction process is much longer than the cloning-free method. Variations on library construction have been produced by
649:
Chen, X.; Xu, H.; Yuan, P.; Fang, F.; Huss, M.; Vega, V. B.; Wong, E.; Orlov, Y. L.; Zhang, W.; Jiang, J.; Loh, Y. H.; Yeo, H. C.; Yeo, Z. X.; Narang, V.; Govindarajan, K. R.; Leong, B.; Shahab, A.; Ruan, Y.; Bourque, G.; Sung, W. K.; Clarke, N. D.; Wei, C. L.; Ng, H. H. (2008).
296:
procedure is used construct the cDNA library before generating the PETs, cDNAs that are difficult to clone (as a result of long transcripts) would have lower coverage. Similarly, transcripts (or transcript isoforms) with low expression levels would likely be under-represented as
240:
through RNA-PET in that the paired tags map to different regions in the genome. However, ChIA-PET involves artificial ligations between different DNA fragments located at different genomic regions, rather than naturally occurring fusion between two genomic regions as in
43:
by consisting of a short 5' linker sequence, a short 5' sequence tag, a short 3' sequence tag, and a short 3' linker sequence. It was shown conceptually that 13 base pairs are sufficient to map tags uniquely. However, longer sequences are more practical for mapping
801:
Ng, P.; Wei, C. L.; Sung, W. K.; Chiu, K. P.; Lipovich, L.; Ang, C. C.; Gupta, S.; Shahab, A.; Ridwan, A.; Wong, C. H.; Liu, E. T.; Ruan, Y. (2005). "Gene identification signature (GIS) analysis for transcriptome characterization and genome annotation".
845:
Ruan, Y.; Ooi, H. S.; Choo, S. W.; Chiu, K. P.; Zhao, X. D.; Srinivasan, K. G.; Yao, F.; Choo, C. Y.; Liu, J.; Ariyaratne, P.; Bin, W. G.; Kuznetsov, V. A.; Shahab, A.; Sung, W. K.; Bourque, G.; Palanisamy, N.; Wei, C. L. (2007).
200:) and PET is used to detect regions of DNA bound by a protein of interest. ChIP-PET has the advantage over single read sequencing by reducing ambiguity of the reads generated. The advantage over chip hybridization (
35:, therefore making the sequence of the DNA in between them available upon search (if full-genome sequence data is available) or upon further sequencing (since tag sites are unique enough to serve as
89:. The cloning sites are flanked with adaptor sequences that contain restriction sites for endonucleases (discussed below). Inserts are ligated to the plasmid vectors and individual vectors are then
232:
cells. ChIA-PET is an unbiased way to analyze interactions and higher-order chromatin structures because it can detect interactions between unknown DNA elements. In contrast,
39:
annealing sites). Paired-end tags (PET) exist in PET libraries with the intervening DNA absent, that is, a PET "represents" a larger fragment of genomic or
263:, but further experiment is needed to distinguish between them. Other methods of finding the boundaries of transcripts include the single-tag strategies
848:"Fusion transcripts and transcribed retrotransposed loci discovered through comprehensive transcriptome analysis using Paired-End diTags (PETs)"
496:"Sequence and structural variation in a human genome uncovered by short-read, massively parallel ligation sequencing using two-base encoding"
192:
435:
Matsumura, H.; Reich, S.; Ito, A.; Saitoh, H.; Kamoun, S.; Winter, P.; Kahl, G.; Reuter, M.; Kruger, D. H.; Terauchi, R. (2003).
910:
156:: Because PET represent connectivity between the tags, the use of PET in genome re-sequencing has advantages over the use of
204:) is that hybridization tiling arrays do not have the statistical sensitivity that sequence reads have. However, ChIP-PET,
236:
methods are used to detect interactions involving a specific target region in the genome. ChIA-PET is similar to finding
268:
161:
699:
Wu, J.; Smith, L. T.; Plass, C.; Huang, T. H. (2006). "ChIP-chip comes of age for genome-wide functional analysis".
220:
long-range interactions between DNA elements bound by protein factors. The first ChIA-PET was developed by
Fullwood
543:
Barski, A.; Cuddapah, S.; Cui, K.; Roh, T. Y.; Schones, D. E.; Wang, Z.; Wei, G.; Chepelev, I.; Zhao, K. (2007).
233:
216:: The application of PET sequencing on chromatin interaction analysis. It is a genome-wide strategy for finding
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388:"MmeI: A minimal Type II restriction-modification system that only modifies one DNA strand for host protection"
56:
method, which has a longer read length. Since the tag sequences are short, individual PETs are well suited for
264:
652:"Integration of external signaling pathways with the core transcriptional network in embryonic stem cells"
112:
99:
57:
284:. The major limitation of RNA-PET is the lack of information regarding the organization of the internal
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36:
185:
124:
90:
77:
PET libraries are typically prepared in two general methods: cloning based and cloning-free based.
915:
275:, with the CAGE and 5’ SAGE defining the transcription start sites and the 3’ SAGE defining the
339:"Next-generation DNA sequencing of paired-end tags (PET) for transcriptome and genome analyses"
181:
102:
technique, PET sequences can be left singular, dimerized, or concatenated into long chains.
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8:
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fragment which are unique enough that they (theoretically) exist together only once in a
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19:(sometimes "Paired-End diTags", or simply "ditags") are the short sequences at the
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Proceedings of the
National Academy of Sciences of the United States of America
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Fragmented genomic DNA or complementary DNA (cDNA) of interest is cloned into
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437:"Gene expression analysis of plant host-pathogen interactions by SuperSAGE"
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expensive than RNA-PET sequencing, especially for characterizing the whole
256:
237:
201:
511:
354:
224:. (2009) to generate a map of the interactions between chromatin bound by
403:
136:
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sequences unique to the clone are now paired together. Depending on the
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545:"High-resolution profiling of histone methylations in the human genome"
123:
Unlike other endonucleases, the MmeI (type IIS) and EcoP15I (type III)
815:
272:
24:
20:
73:
Workflow of
Cloning and Cloning-free based PET library construction.
212:
293:
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of transcripts. Therefore, RNA-PET is not suitable for detecting
148:
Example of alternative transcript structures detected by RNA-PET.
386:
Morgan, R. D.; Bhatia, T. K.; Lovasco, L.; Davis, T. B. (2008).
741:"An oestrogen-receptor-alpha-bound human chromatin interactome"
32:
592:
Johnson, D. S.; Mortazavi, A.; Myers, R. M.; Wold, B. (2007).
493:
69:
285:
40:
337:
Fullwood, M. J.; Wei, C. L.; Liu, E. T.; Ruan, Y. (2009).
144:
594:"Genome-wide mapping of in vivo protein-DNA interactions"
591:
28:
385:
434:
255:
sites of genes. RNA-PET could also be used to detect
196:: The combined use of chromatin immunoprecipitation (
140:
Example of PET detection of deletions and insertions.
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115:companies to suit their respective technologies.
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64:
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208:and ChIP-chip have all been highly successful.
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247:: This application is used for studying the
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228:(ER-α) in oestrogen-treated human breast
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739:Fullwood, M. J.; et al. (2009).
494:McKernan, K. J.; et al. (2009).
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13:
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128:ligated to them, producing PETs.
166:double-barrel shotgun sequencing
118:
80:
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160:. This application is called
1:
713:10.1158/0008-5472.CAN-06-0276
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911:Molecular biology techniques
65:Constructing the PET library
7:
10:
932:
669:10.1016/j.cell.2008.04.043
562:10.1016/j.cell.2007.05.009
113:next-generation sequencing
100:next-generation sequencing
61:end of the DNA fragment.
58:next-generation sequencing
125:restriction endonucleases
164:, known colloquially as
619:10.1126/science.1141319
462:10.1073/pnas.2536670100
162:pairwise end sequencing
392:Nucleic Acids Research
292:. In addition, if the
271:, and the most recent
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141:
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906:Laboratory techniques
512:10.1101/gr.091868.109
355:10.1101/gr.074906.107
147:
139:
72:
17:Paired-end tags (PET)
290:alternative splicing
226:oestrogen receptor α
765:10.1038/nature08497
757:2009Natur.462...58F
610:2007Sci...316.1497J
604:(5830): 1497–1902.
453:2003PNAS..10015718M
447:(26): 15718–15723.
864:10.1101/gr.6018607
404:10.1093/nar/gkn711
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106:Cloning-free based
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707:(14): 6899–7702.
398:(20): 6558–6570.
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132:PET applications
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257:fusion genes
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238:fusion genes
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178:duplications
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158:single reads
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91:transformed
895:Categories
302:References
182:inversions
170:insertions
273:SuperSAGE
234:3C and 4C
202:ChIP-Chip
174:deletions
882:17568001
832:14288213
824:15782207
783:19890323
721:16849531
678:18555785
628:17540862
571:17512414
530:19546169
481:14676315
422:18931376
373:19339662
241:RNA-PET.
213:ChIA-PET
206:ChIP-Seq
193:ChIP-PET
873:1891342
774:2774924
753:Bibcode
686:1768190
606:Bibcode
598:Science
579:6326093
521:2752135
449:Bibcode
413:2582602
364:3807531
294:cloning
245:RNA-PET
218:de novo
154:DNA-PET
95:E. coli
25:3' ends
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222:et al.
54:Sanger
37:primer
33:genome
828:S2CID
682:S2CID
632:S2CID
575:S2CID
297:well.
286:exons
93:into
46:reads
27:of a
878:PMID
820:PMID
779:PMID
717:PMID
674:PMID
656:Cell
624:PMID
567:PMID
549:Cell
526:PMID
477:PMID
418:PMID
369:PMID
269:SAGE
265:CAGE
259:and
198:ChIP
41:cDNA
23:and
868:PMC
860:doi
812:doi
769:PMC
761:doi
749:462
709:doi
664:doi
660:133
614:doi
602:316
557:doi
553:129
516:PMC
508:doi
467:PMC
457:doi
445:100
408:PMC
400:doi
359:PMC
351:doi
29:DNA
897::
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