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Assessing long-distance RNA sequence connectivity via RNA-templated DNA-DNA ligation.

Roy CK, Olson S, Graveley BR, Zamore PD, Moore MJ - Elife (2015)

Bottom Line: Multiple sites of alternative splicing within a single gene exponentially increase the number of possible spliced isoforms, with most human genes currently estimated to express at least ten.To understand the mechanisms underlying these complex isoform expression patterns, methods are needed that faithfully maintain long-range exon connectivity information in individual RNA molecules.Using this assay, we test proposed coordination between distant sites of alternative exon utilization in mouse Fn1, and we characterize the extraordinary exon diversity of Drosophila melanogaster Dscam1.

View Article: PubMed Central - PubMed

Affiliation: RNA Therapeutics Institute, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, United States.

ABSTRACT
Many RNAs, including pre-mRNAs and long non-coding RNAs, can be thousands of nucleotides long and undergo complex post-transcriptional processing. Multiple sites of alternative splicing within a single gene exponentially increase the number of possible spliced isoforms, with most human genes currently estimated to express at least ten. To understand the mechanisms underlying these complex isoform expression patterns, methods are needed that faithfully maintain long-range exon connectivity information in individual RNA molecules. In this study, we describe SeqZip, a methodology that uses RNA-templated DNA-DNA ligation to retain and compress connectivity between distant sequences within single RNA molecules. Using this assay, we test proposed coordination between distant sites of alternative exon utilization in mouse Fn1, and we characterize the extraordinary exon diversity of Drosophila melanogaster Dscam1.

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Other proposed uses of SeqZip.Shown are various uses of SeqZip toward multi-site sequence investigation of RNA. ‘Product Length Adjustment’ has applications similar to those shown in Figure 3E, where isoform discrimination solely on the basis of size separation of RT-PCR products would be ambiguous; with SeqZip, the lengths of individual products can be adjusted through ligamer design. ‘RNA barcoding’ depicts the introduction of randomized rather than static barcodes, allowing for molecular indexing or amplification bias estimation. ‘Quantify RNA-integrity’ relies on the requirement of molecular continuity between sites of ligamer hybridization in order to obtain a SeqZip product (check mark). If the intervening sequences are not intact, no product is obtained (X). Thus, SeqZip can be used to monitor the integrity of long RNAs. ‘Multi-site SNP detection’ is described in the ‘Discussion’ section ‘SeqZip uses and limitations’. The panel depicting ‘Introduction of destruction sequences’ illustrates how short DNA oligos targeting ligamer-specific barcodes between hybridization regions (in this case ‘B’) could be useful in the selective cleavage and destruction of particular ligation products. In the example shown, the ABC ligamer product would be cleaved with a restriction enzyme targeting the double-stranded oligo:barcode, while DEF would be left intact for downstream applications. ‘Sequence discovery using combined SeqZip and Reverse Transcription’ illustrates 5′ end sequence discovery using Cap Analysis of Gene Expression combined with SeqZip ligamers. This allows one to investigate novel 5′ end sequence connections to distant 3′ sequences. ‘Multi-site AS QPCR analysis’ is also described in the ‘Discussion’ section ‘SeqZip uses and limitations’. The essential benefit over a conventional QPCR workflow is that SeqZip compresses distant sequences into a QPCR-friendly amplicon size and reduces the number of required primers.DOI:http://dx.doi.org/10.7554/eLife.03700.005
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fig1s2: Other proposed uses of SeqZip.Shown are various uses of SeqZip toward multi-site sequence investigation of RNA. ‘Product Length Adjustment’ has applications similar to those shown in Figure 3E, where isoform discrimination solely on the basis of size separation of RT-PCR products would be ambiguous; with SeqZip, the lengths of individual products can be adjusted through ligamer design. ‘RNA barcoding’ depicts the introduction of randomized rather than static barcodes, allowing for molecular indexing or amplification bias estimation. ‘Quantify RNA-integrity’ relies on the requirement of molecular continuity between sites of ligamer hybridization in order to obtain a SeqZip product (check mark). If the intervening sequences are not intact, no product is obtained (X). Thus, SeqZip can be used to monitor the integrity of long RNAs. ‘Multi-site SNP detection’ is described in the ‘Discussion’ section ‘SeqZip uses and limitations’. The panel depicting ‘Introduction of destruction sequences’ illustrates how short DNA oligos targeting ligamer-specific barcodes between hybridization regions (in this case ‘B’) could be useful in the selective cleavage and destruction of particular ligation products. In the example shown, the ABC ligamer product would be cleaved with a restriction enzyme targeting the double-stranded oligo:barcode, while DEF would be left intact for downstream applications. ‘Sequence discovery using combined SeqZip and Reverse Transcription’ illustrates 5′ end sequence discovery using Cap Analysis of Gene Expression combined with SeqZip ligamers. This allows one to investigate novel 5′ end sequence connections to distant 3′ sequences. ‘Multi-site AS QPCR analysis’ is also described in the ‘Discussion’ section ‘SeqZip uses and limitations’. The essential benefit over a conventional QPCR workflow is that SeqZip compresses distant sequences into a QPCR-friendly amplicon size and reduces the number of required primers.DOI:http://dx.doi.org/10.7554/eLife.03700.005

Mentions: One potential future application of SeqZip is the detection of multiple single-nucleotide polymorphisms (SNPs) on a single molecule of a long RNA. By placing the ligation sites over each SNP, one could take advantage of the requirement by Rnl2 for complete complementarity at a ligation junction; mismatches would inhibit efficient ligamer joining (Landegren et al., 1988; Chauleau and Shuman, 2013). Further, any sequence can be placed in between the two regions of target complementarity within each ligamer. Therefore, sequences for custom priming, restriction digestion, recombination, etc, can be introduced, allowing for quantification or subsequent manipulation of ligation products. Analysis of ligation products can even be multiplexed, allowing for simultaneous generation and analysis using internal controls. These applications and others are shown in Figure 1—figure supplement 2.


Assessing long-distance RNA sequence connectivity via RNA-templated DNA-DNA ligation.

Roy CK, Olson S, Graveley BR, Zamore PD, Moore MJ - Elife (2015)

Other proposed uses of SeqZip.Shown are various uses of SeqZip toward multi-site sequence investigation of RNA. ‘Product Length Adjustment’ has applications similar to those shown in Figure 3E, where isoform discrimination solely on the basis of size separation of RT-PCR products would be ambiguous; with SeqZip, the lengths of individual products can be adjusted through ligamer design. ‘RNA barcoding’ depicts the introduction of randomized rather than static barcodes, allowing for molecular indexing or amplification bias estimation. ‘Quantify RNA-integrity’ relies on the requirement of molecular continuity between sites of ligamer hybridization in order to obtain a SeqZip product (check mark). If the intervening sequences are not intact, no product is obtained (X). Thus, SeqZip can be used to monitor the integrity of long RNAs. ‘Multi-site SNP detection’ is described in the ‘Discussion’ section ‘SeqZip uses and limitations’. The panel depicting ‘Introduction of destruction sequences’ illustrates how short DNA oligos targeting ligamer-specific barcodes between hybridization regions (in this case ‘B’) could be useful in the selective cleavage and destruction of particular ligation products. In the example shown, the ABC ligamer product would be cleaved with a restriction enzyme targeting the double-stranded oligo:barcode, while DEF would be left intact for downstream applications. ‘Sequence discovery using combined SeqZip and Reverse Transcription’ illustrates 5′ end sequence discovery using Cap Analysis of Gene Expression combined with SeqZip ligamers. This allows one to investigate novel 5′ end sequence connections to distant 3′ sequences. ‘Multi-site AS QPCR analysis’ is also described in the ‘Discussion’ section ‘SeqZip uses and limitations’. The essential benefit over a conventional QPCR workflow is that SeqZip compresses distant sequences into a QPCR-friendly amplicon size and reduces the number of required primers.DOI:http://dx.doi.org/10.7554/eLife.03700.005
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4442144&req=5

fig1s2: Other proposed uses of SeqZip.Shown are various uses of SeqZip toward multi-site sequence investigation of RNA. ‘Product Length Adjustment’ has applications similar to those shown in Figure 3E, where isoform discrimination solely on the basis of size separation of RT-PCR products would be ambiguous; with SeqZip, the lengths of individual products can be adjusted through ligamer design. ‘RNA barcoding’ depicts the introduction of randomized rather than static barcodes, allowing for molecular indexing or amplification bias estimation. ‘Quantify RNA-integrity’ relies on the requirement of molecular continuity between sites of ligamer hybridization in order to obtain a SeqZip product (check mark). If the intervening sequences are not intact, no product is obtained (X). Thus, SeqZip can be used to monitor the integrity of long RNAs. ‘Multi-site SNP detection’ is described in the ‘Discussion’ section ‘SeqZip uses and limitations’. The panel depicting ‘Introduction of destruction sequences’ illustrates how short DNA oligos targeting ligamer-specific barcodes between hybridization regions (in this case ‘B’) could be useful in the selective cleavage and destruction of particular ligation products. In the example shown, the ABC ligamer product would be cleaved with a restriction enzyme targeting the double-stranded oligo:barcode, while DEF would be left intact for downstream applications. ‘Sequence discovery using combined SeqZip and Reverse Transcription’ illustrates 5′ end sequence discovery using Cap Analysis of Gene Expression combined with SeqZip ligamers. This allows one to investigate novel 5′ end sequence connections to distant 3′ sequences. ‘Multi-site AS QPCR analysis’ is also described in the ‘Discussion’ section ‘SeqZip uses and limitations’. The essential benefit over a conventional QPCR workflow is that SeqZip compresses distant sequences into a QPCR-friendly amplicon size and reduces the number of required primers.DOI:http://dx.doi.org/10.7554/eLife.03700.005
Mentions: One potential future application of SeqZip is the detection of multiple single-nucleotide polymorphisms (SNPs) on a single molecule of a long RNA. By placing the ligation sites over each SNP, one could take advantage of the requirement by Rnl2 for complete complementarity at a ligation junction; mismatches would inhibit efficient ligamer joining (Landegren et al., 1988; Chauleau and Shuman, 2013). Further, any sequence can be placed in between the two regions of target complementarity within each ligamer. Therefore, sequences for custom priming, restriction digestion, recombination, etc, can be introduced, allowing for quantification or subsequent manipulation of ligation products. Analysis of ligation products can even be multiplexed, allowing for simultaneous generation and analysis using internal controls. These applications and others are shown in Figure 1—figure supplement 2.

Bottom Line: Multiple sites of alternative splicing within a single gene exponentially increase the number of possible spliced isoforms, with most human genes currently estimated to express at least ten.To understand the mechanisms underlying these complex isoform expression patterns, methods are needed that faithfully maintain long-range exon connectivity information in individual RNA molecules.Using this assay, we test proposed coordination between distant sites of alternative exon utilization in mouse Fn1, and we characterize the extraordinary exon diversity of Drosophila melanogaster Dscam1.

View Article: PubMed Central - PubMed

Affiliation: RNA Therapeutics Institute, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, United States.

ABSTRACT
Many RNAs, including pre-mRNAs and long non-coding RNAs, can be thousands of nucleotides long and undergo complex post-transcriptional processing. Multiple sites of alternative splicing within a single gene exponentially increase the number of possible spliced isoforms, with most human genes currently estimated to express at least ten. To understand the mechanisms underlying these complex isoform expression patterns, methods are needed that faithfully maintain long-range exon connectivity information in individual RNA molecules. In this study, we describe SeqZip, a methodology that uses RNA-templated DNA-DNA ligation to retain and compress connectivity between distant sequences within single RNA molecules. Using this assay, we test proposed coordination between distant sites of alternative exon utilization in mouse Fn1, and we characterize the extraordinary exon diversity of Drosophila melanogaster Dscam1.

Show MeSH
Related in: MedlinePlus