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A method for generating highly multiplexed ChIP-seq libraries.

Ford E, Nikopoulou C, Kokkalis A, Thanos D - BMC Res Notes (2014)

Bottom Line: However, current barcoding strategies impose significant limitations and/or technical barriers in their implementation for ChIP-sequencing.Converting Y-shaped sequencing adapters to double stranded DNA prior to agarose gel size selection reduces adapter dimer contamination and quantitating the number of cycles required for amplification of the library with qPCR prior to library amplification eliminates library over-amplification.We describe an efficient and cost effective method for making barcoded ChIP-seq libraries for sequencing on the Illumina platform.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biomedical Research Foundation, Academy of Athens, 4 Soranou Efesiou Street, Athens 11527, Greece. thanos@bioacademy.gr.

ABSTRACT

Background: The barcoding of next generation sequencing libraries has become an essential part of the experimental design. Barcoding not only allows the sequencing of more than one sample per lane, but also reduces technical bias. However, current barcoding strategies impose significant limitations and/or technical barriers in their implementation for ChIP-sequencing.

Findings: Converting Y-shaped sequencing adapters to double stranded DNA prior to agarose gel size selection reduces adapter dimer contamination and quantitating the number of cycles required for amplification of the library with qPCR prior to library amplification eliminates library over-amplification.

Conclusions: We describe an efficient and cost effective method for making barcoded ChIP-seq libraries for sequencing on the Illumina platform.

Show MeSH
Validation of the ChIP-seq library construction method by ChIP-seq against macroH2A1.2.(A) Bioanalyzer 2100 gel image showing 1 ul of each library run on DNA 1000 chip (B) Validation of several macroH2A1.2 peaks by qPCR at the respective loci. Three loci not bound by macroH2A1 are on the left side (red) and eight loci identified as macroH2A1 peaks are on the right side (green). Loci are named according to their location on the mm9 mouse genome build. (C) A UCSC genome browser screen shot of representative macroH2A1.2 peaks. Input DNA is colored in blue and the two anti-macroH2A1.2 ChIP libraries A and B in red.
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Figure 2: Validation of the ChIP-seq library construction method by ChIP-seq against macroH2A1.2.(A) Bioanalyzer 2100 gel image showing 1 ul of each library run on DNA 1000 chip (B) Validation of several macroH2A1.2 peaks by qPCR at the respective loci. Three loci not bound by macroH2A1 are on the left side (red) and eight loci identified as macroH2A1 peaks are on the right side (green). Loci are named according to their location on the mm9 mouse genome build. (C) A UCSC genome browser screen shot of representative macroH2A1.2 peaks. Input DNA is colored in blue and the two anti-macroH2A1.2 ChIP libraries A and B in red.

Mentions: To demonstrate the robustness of our library preparation protocol we performed ChIP against the histone variant macroH2A1 in mouse embryonic fibroblasts (MEFs). MacroH2A1 is a unique histone variant, in that in addition to the canonical histone H2A domain, it also contains a relatively large (30 kD) amino-terminal ‘macro’ domain. While macroH2A1 is generally associated with gene repression, it is also required for the activation of a subset of genes [8]. The alternative use of a 64 bp or 73 bp mutually exclusive exon results in the production of two distinct proteins, macroH2A1.1 and macroH2A1.2, respectively [9]. While macroH2A1.1 efficiently binds the NAD+ metabolite ADP-ribose, macroH2A1.2 does not [10]. We prepared Illumina libraries from immunoprecipitated material using antibodies targeted to macroH2A1.2 as well as input DNA (for a detailed protocol see Additional file 1). Five ChIP-seq libraries were made, two from the macroH2A1.2 immunoprecipitated DNA (macroH2A1.2 library A and macroH2A1.2 library B), two from the input DNA (input library A and input library B) and one from input DNA in which the pre-agaros gel PCR step was omitted. The libraries were amplified with 12 cycles of PCR (including the pre-size selection PCR cycles) and visualized on the Agilent Bioanalyzer (Figure 2A) before being sequenced in one lane of the HiSeq2000. In total, 76 million reads were obtained and demultiplexed with Illumina’s Casava data analysis pipeline. 24 million, 15 million, 21 million and 15 million reads for macroH2A1.2 library A, macroH2A1.2 library B, input library A and input library B respectively, were mapped to the mouse genome build mm9 with the Bowtie short read alignment software. MacroH2A1.2 enriched regions were identified with the MACS software package [11] and the data sets were uploaded onto the UCSC genome browser. In total we identified 33 peaks in macroH2A1.2 library A. Every peak identified in macroH2A library A was also a peak in macroH2A library B, demonstrating the reproducibility of the library construction protocol. An example peak is shown in Figure 2C as a UCSC genome browser screenshot. A few of the identified peaks were validated by ChIP-qPCR (Figure 2B) further supporting the specificity and the effectiveness of the method. Adapter sequences represented less than 1% of sequences, which highlights the effectiveness and necessity to convert the Y-shaped adapters to double-stranded DNA prior to agarose gel size selection.


A method for generating highly multiplexed ChIP-seq libraries.

Ford E, Nikopoulou C, Kokkalis A, Thanos D - BMC Res Notes (2014)

Validation of the ChIP-seq library construction method by ChIP-seq against macroH2A1.2.(A) Bioanalyzer 2100 gel image showing 1 ul of each library run on DNA 1000 chip (B) Validation of several macroH2A1.2 peaks by qPCR at the respective loci. Three loci not bound by macroH2A1 are on the left side (red) and eight loci identified as macroH2A1 peaks are on the right side (green). Loci are named according to their location on the mm9 mouse genome build. (C) A UCSC genome browser screen shot of representative macroH2A1.2 peaks. Input DNA is colored in blue and the two anti-macroH2A1.2 ChIP libraries A and B in red.
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Related In: Results  -  Collection

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Figure 2: Validation of the ChIP-seq library construction method by ChIP-seq against macroH2A1.2.(A) Bioanalyzer 2100 gel image showing 1 ul of each library run on DNA 1000 chip (B) Validation of several macroH2A1.2 peaks by qPCR at the respective loci. Three loci not bound by macroH2A1 are on the left side (red) and eight loci identified as macroH2A1 peaks are on the right side (green). Loci are named according to their location on the mm9 mouse genome build. (C) A UCSC genome browser screen shot of representative macroH2A1.2 peaks. Input DNA is colored in blue and the two anti-macroH2A1.2 ChIP libraries A and B in red.
Mentions: To demonstrate the robustness of our library preparation protocol we performed ChIP against the histone variant macroH2A1 in mouse embryonic fibroblasts (MEFs). MacroH2A1 is a unique histone variant, in that in addition to the canonical histone H2A domain, it also contains a relatively large (30 kD) amino-terminal ‘macro’ domain. While macroH2A1 is generally associated with gene repression, it is also required for the activation of a subset of genes [8]. The alternative use of a 64 bp or 73 bp mutually exclusive exon results in the production of two distinct proteins, macroH2A1.1 and macroH2A1.2, respectively [9]. While macroH2A1.1 efficiently binds the NAD+ metabolite ADP-ribose, macroH2A1.2 does not [10]. We prepared Illumina libraries from immunoprecipitated material using antibodies targeted to macroH2A1.2 as well as input DNA (for a detailed protocol see Additional file 1). Five ChIP-seq libraries were made, two from the macroH2A1.2 immunoprecipitated DNA (macroH2A1.2 library A and macroH2A1.2 library B), two from the input DNA (input library A and input library B) and one from input DNA in which the pre-agaros gel PCR step was omitted. The libraries were amplified with 12 cycles of PCR (including the pre-size selection PCR cycles) and visualized on the Agilent Bioanalyzer (Figure 2A) before being sequenced in one lane of the HiSeq2000. In total, 76 million reads were obtained and demultiplexed with Illumina’s Casava data analysis pipeline. 24 million, 15 million, 21 million and 15 million reads for macroH2A1.2 library A, macroH2A1.2 library B, input library A and input library B respectively, were mapped to the mouse genome build mm9 with the Bowtie short read alignment software. MacroH2A1.2 enriched regions were identified with the MACS software package [11] and the data sets were uploaded onto the UCSC genome browser. In total we identified 33 peaks in macroH2A1.2 library A. Every peak identified in macroH2A library A was also a peak in macroH2A library B, demonstrating the reproducibility of the library construction protocol. An example peak is shown in Figure 2C as a UCSC genome browser screenshot. A few of the identified peaks were validated by ChIP-qPCR (Figure 2B) further supporting the specificity and the effectiveness of the method. Adapter sequences represented less than 1% of sequences, which highlights the effectiveness and necessity to convert the Y-shaped adapters to double-stranded DNA prior to agarose gel size selection.

Bottom Line: However, current barcoding strategies impose significant limitations and/or technical barriers in their implementation for ChIP-sequencing.Converting Y-shaped sequencing adapters to double stranded DNA prior to agarose gel size selection reduces adapter dimer contamination and quantitating the number of cycles required for amplification of the library with qPCR prior to library amplification eliminates library over-amplification.We describe an efficient and cost effective method for making barcoded ChIP-seq libraries for sequencing on the Illumina platform.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biomedical Research Foundation, Academy of Athens, 4 Soranou Efesiou Street, Athens 11527, Greece. thanos@bioacademy.gr.

ABSTRACT

Background: The barcoding of next generation sequencing libraries has become an essential part of the experimental design. Barcoding not only allows the sequencing of more than one sample per lane, but also reduces technical bias. However, current barcoding strategies impose significant limitations and/or technical barriers in their implementation for ChIP-sequencing.

Findings: Converting Y-shaped sequencing adapters to double stranded DNA prior to agarose gel size selection reduces adapter dimer contamination and quantitating the number of cycles required for amplification of the library with qPCR prior to library amplification eliminates library over-amplification.

Conclusions: We describe an efficient and cost effective method for making barcoded ChIP-seq libraries for sequencing on the Illumina platform.

Show MeSH