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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.

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A schematic representation of the method for making barcoded ChIP-seq libraries. An asterisk denotes novel steps in the protocol. (A) End-repair. (B) A-tailing. (C) Adapter ligation. (D) Conversion of Y-shaped DNA to double-stranded DNA. (E) Size selection. (F) Cycle quantitation. (G) PCR amplification.
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Figure 1: A schematic representation of the method for making barcoded ChIP-seq libraries. An asterisk denotes novel steps in the protocol. (A) End-repair. (B) A-tailing. (C) Adapter ligation. (D) Conversion of Y-shaped DNA to double-stranded DNA. (E) Size selection. (F) Cycle quantitation. (G) PCR amplification.

Mentions: Here, we describe a method that bypasses the problems associated with size selection. Specifically, compared to the two methods discussed, we perform five cycles of PCR prior to size selection on an agarose gel to convert the Y-shaped DNA to double-stranded DNA, so that both the adapter-dimers and adapter-ligated immunoprecipitated DNA run true to their size during agarose gel electrophoresis and accurate size selection can be performed. This step also results in a modest amplification of the immunoprecipitated DNA prior to agarose gel size selection, which increases the yield of an otherwise inefficient step in the library preparation protocol. In addition, we also include a method to accurately quantitate the number of cycles required for PCR amplification of the library, which reduces biases caused by over-amplification [6]. Library amplification is performed using Kapa HiFi polymerase, which is more efficient and produces less bias than Phusion polymerase [7]. Finally, we have reduced the amount of reagents required for library construction making library construction more affordable, which is especially important as sequencing costs drop.A schematic step-wise representation of the method is illustrated in Figure 1. Briefly, a minimum of 2 ng of immunoprecipitated DNA is treated with T4 DNA polymerase, Klenow fragment and T4 polynucleotide kinase to blunt the DNA ends and add 3’ phosphates (Figure 1A). Next, single 5’ adenine overhangs are generated by incubating the DNA with Klenow exo-minus and dATP (Figure 1B). Barcoded Y-shaped oligonucleotide adapters are ligated to the DNA (Figure 1C). The DNA molecules are then converted to double-stranded DNA with five cycles of PCR (Figure 1D), so that the DNA molecules run predictably during the subsequent size selection by agarose gel electrophoresis (Figure 1E). The precise number of PCR cycles required for amplification is determined by qPCR (Figure 1F) and finally the library is amplified by PCR accordingly (Figure 1G).


A method for generating highly multiplexed ChIP-seq libraries.

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

A schematic representation of the method for making barcoded ChIP-seq libraries. An asterisk denotes novel steps in the protocol. (A) End-repair. (B) A-tailing. (C) Adapter ligation. (D) Conversion of Y-shaped DNA to double-stranded DNA. (E) Size selection. (F) Cycle quantitation. (G) PCR amplification.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4048252&req=5

Figure 1: A schematic representation of the method for making barcoded ChIP-seq libraries. An asterisk denotes novel steps in the protocol. (A) End-repair. (B) A-tailing. (C) Adapter ligation. (D) Conversion of Y-shaped DNA to double-stranded DNA. (E) Size selection. (F) Cycle quantitation. (G) PCR amplification.
Mentions: Here, we describe a method that bypasses the problems associated with size selection. Specifically, compared to the two methods discussed, we perform five cycles of PCR prior to size selection on an agarose gel to convert the Y-shaped DNA to double-stranded DNA, so that both the adapter-dimers and adapter-ligated immunoprecipitated DNA run true to their size during agarose gel electrophoresis and accurate size selection can be performed. This step also results in a modest amplification of the immunoprecipitated DNA prior to agarose gel size selection, which increases the yield of an otherwise inefficient step in the library preparation protocol. In addition, we also include a method to accurately quantitate the number of cycles required for PCR amplification of the library, which reduces biases caused by over-amplification [6]. Library amplification is performed using Kapa HiFi polymerase, which is more efficient and produces less bias than Phusion polymerase [7]. Finally, we have reduced the amount of reagents required for library construction making library construction more affordable, which is especially important as sequencing costs drop.A schematic step-wise representation of the method is illustrated in Figure 1. Briefly, a minimum of 2 ng of immunoprecipitated DNA is treated with T4 DNA polymerase, Klenow fragment and T4 polynucleotide kinase to blunt the DNA ends and add 3’ phosphates (Figure 1A). Next, single 5’ adenine overhangs are generated by incubating the DNA with Klenow exo-minus and dATP (Figure 1B). Barcoded Y-shaped oligonucleotide adapters are ligated to the DNA (Figure 1C). The DNA molecules are then converted to double-stranded DNA with five cycles of PCR (Figure 1D), so that the DNA molecules run predictably during the subsequent size selection by agarose gel electrophoresis (Figure 1E). The precise number of PCR cycles required for amplification is determined by qPCR (Figure 1F) and finally the library is amplified by PCR accordingly (Figure 1G).

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