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Serial number tagging reveals a prominent sequence preference of retrotransposon integration.

Chatterjee AG, Esnault C, Guo Y, Hung S, McQueen PG, Levin HL - Nucleic Acids Res. (2014)

Bottom Line: To address this problem we developed the serial number system, a TE tagging method that measures the frequency of integration at single nucleotide positions.We sequenced 1 million insertions of retrotransposon Tf1 in the genome of Schizosaccharomyces pombe and obtained the first profile of integration with frequencies for each individual position.Integration levels at individual nucleotides varied over two orders of magnitude and revealed that sequence recognition plays a key role in positioning integration.

View Article: PubMed Central - PubMed

Affiliation: Section on Eukaryotic Transposable Elements, Program in Cellular Regulation and Metabolism, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

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The serial number system quantifies the number of independent insertions at single nucleotide positions. (A) Two hypothetical promoters each have insertions at three different positions. However, one promoter (right) could have many more insertions at each site than the other. Because the insertion libraries are created by PCR duplicate sequence reads are typically discarded. As a result, two promoters could have very different amounts of integration but be reported to have the same amount of integration. (B) (i) The serial number system was created in Tf1 by inserting 8 bp of random sequence in the U5 region of the 5′ LTR (red stripe). Tf1 mRNA was expressed from the nmt1 promoter within a library of plasmids. (ii) The library of expression plasmids with the serial number sequences were introduced into S. pombe (iii) and the expression of Tf1 was induced (iv). (v) Genomic DNA from cells with Tf1 was ligated to linkers (red) and the insertions were amplified by PCR. The products were sequenced with Illumina technology. The sequence primer read across the serial numbers and into the sequence of the insertion sites (blue).
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Figure 1: The serial number system quantifies the number of independent insertions at single nucleotide positions. (A) Two hypothetical promoters each have insertions at three different positions. However, one promoter (right) could have many more insertions at each site than the other. Because the insertion libraries are created by PCR duplicate sequence reads are typically discarded. As a result, two promoters could have very different amounts of integration but be reported to have the same amount of integration. (B) (i) The serial number system was created in Tf1 by inserting 8 bp of random sequence in the U5 region of the 5′ LTR (red stripe). Tf1 mRNA was expressed from the nmt1 promoter within a library of plasmids. (ii) The library of expression plasmids with the serial number sequences were introduced into S. pombe (iii) and the expression of Tf1 was induced (iv). (v) Genomic DNA from cells with Tf1 was ligated to linkers (red) and the insertions were amplified by PCR. The products were sequenced with Illumina technology. The sequence primer read across the serial numbers and into the sequence of the insertion sites (blue).

Mentions: To study genome-wide integration, a copy of Tf1 marked with neo (Tf1-neo) is expressed in S. pombe and cells with insertions are selected on media containing G418 (18,19). High-throughput sequencing of Tf1-neo integration sites relies on amplifying insertions with ligation-mediated PCR (4). The previous study that demonstrated Tf1-neo integrates into pol II transcribed promoters measured the number of positions with integration. However, the number of insertions at each position could not be determined; the duplicate sequence reads were discarded because those generated by independent integration could not be distinguished from ones resulting from PCR or cell propagation (Figure 1A).


Serial number tagging reveals a prominent sequence preference of retrotransposon integration.

Chatterjee AG, Esnault C, Guo Y, Hung S, McQueen PG, Levin HL - Nucleic Acids Res. (2014)

The serial number system quantifies the number of independent insertions at single nucleotide positions. (A) Two hypothetical promoters each have insertions at three different positions. However, one promoter (right) could have many more insertions at each site than the other. Because the insertion libraries are created by PCR duplicate sequence reads are typically discarded. As a result, two promoters could have very different amounts of integration but be reported to have the same amount of integration. (B) (i) The serial number system was created in Tf1 by inserting 8 bp of random sequence in the U5 region of the 5′ LTR (red stripe). Tf1 mRNA was expressed from the nmt1 promoter within a library of plasmids. (ii) The library of expression plasmids with the serial number sequences were introduced into S. pombe (iii) and the expression of Tf1 was induced (iv). (v) Genomic DNA from cells with Tf1 was ligated to linkers (red) and the insertions were amplified by PCR. The products were sequenced with Illumina technology. The sequence primer read across the serial numbers and into the sequence of the insertion sites (blue).
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Related In: Results  -  Collection

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Figure 1: The serial number system quantifies the number of independent insertions at single nucleotide positions. (A) Two hypothetical promoters each have insertions at three different positions. However, one promoter (right) could have many more insertions at each site than the other. Because the insertion libraries are created by PCR duplicate sequence reads are typically discarded. As a result, two promoters could have very different amounts of integration but be reported to have the same amount of integration. (B) (i) The serial number system was created in Tf1 by inserting 8 bp of random sequence in the U5 region of the 5′ LTR (red stripe). Tf1 mRNA was expressed from the nmt1 promoter within a library of plasmids. (ii) The library of expression plasmids with the serial number sequences were introduced into S. pombe (iii) and the expression of Tf1 was induced (iv). (v) Genomic DNA from cells with Tf1 was ligated to linkers (red) and the insertions were amplified by PCR. The products were sequenced with Illumina technology. The sequence primer read across the serial numbers and into the sequence of the insertion sites (blue).
Mentions: To study genome-wide integration, a copy of Tf1 marked with neo (Tf1-neo) is expressed in S. pombe and cells with insertions are selected on media containing G418 (18,19). High-throughput sequencing of Tf1-neo integration sites relies on amplifying insertions with ligation-mediated PCR (4). The previous study that demonstrated Tf1-neo integrates into pol II transcribed promoters measured the number of positions with integration. However, the number of insertions at each position could not be determined; the duplicate sequence reads were discarded because those generated by independent integration could not be distinguished from ones resulting from PCR or cell propagation (Figure 1A).

Bottom Line: To address this problem we developed the serial number system, a TE tagging method that measures the frequency of integration at single nucleotide positions.We sequenced 1 million insertions of retrotransposon Tf1 in the genome of Schizosaccharomyces pombe and obtained the first profile of integration with frequencies for each individual position.Integration levels at individual nucleotides varied over two orders of magnitude and revealed that sequence recognition plays a key role in positioning integration.

View Article: PubMed Central - PubMed

Affiliation: Section on Eukaryotic Transposable Elements, Program in Cellular Regulation and Metabolism, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

Show MeSH
Related in: MedlinePlus