NEW YORK (GenomeWeb) – Researchers from the Singapore Institute for Clinical Sciences' Agency for Science, Technology and Research (A*STAR) have developed a next-generation sequencing library construction method that can be applied to both ChIP-seq and RNA-seq.
According to the researchers, the main advantages of the method are its versatility and its low input requirement; the team demonstrated that a ChIP-seq library could be made with as little as 25 pg of DNA.
The A*STAR researchers published their method, dubbed TELP, for tailing, extension, ligation, and PCR — the four main steps in the protocol — in Nucleic Acids Research last month.
Xu Feng, the senior author of the study and a PI at the Singapore Institute for Clinical Sciences, told In Sequence that TELP was born out of a desire for a ChIP-seq method that didn't require a large amount of starting material. In addition, he said, the group was "interested in mapping the binding sites of single-stranded DNA binding proteins, but standard [ChIP-seq] cannot process ssDNA."
In the first step, the researchers add a poly-C tail to the 3' end of either ssDNA or dsDNA using terminal deoxynucleotide transferase (TDT). Next, they used the polymerase KAPA-2G for extension, using a biotin-labeled anchor primer to help with downstream purification. After extension, the excess primer was removed with exonuclease digestion so that it would not ligate to the adapter added later. The team found that if they did not remove the excess primer, the anchor primer-adapater dimer "dominated the PCR reaction, leading to the failure of library preparation."
After extension, they used magnetic streptavidin beads to capture the extension products and adapter ligation was performed on the beads. Ligation was performed overnight prior to the final PCR amplification. Index sequences were added in the PCR step to generate sequencing-ready libraries.
To validate the TELP protocol, the team tested it on mouse adipocytes, performing ChIP-seq using antibodies specific to histone H3K4me3, K9ac, K27ac, and K27me3 — markers that are signatures of active promoters in gene regulation. In parallel, the researchers also performed RNA-seq, using the Illumina HiSeq 2000 for both experiments. The epigenomic profiles generated with the TELP library were consistent with standard Illumina ChIP-seq profiles. Comparing the ChIP-seq and RNA-seq results, the researchers found that the histone marks H3KPac, H3K27ac, and H3Kme3 correlated strongly with transcriptional status of the corresponding genes.
Next, they wanted to test how little input DNA could be used and still obtain desirable results. They tested 1ng, 100 pg, and 25 pg and compared those inputs using the TELP method with standard ChIP-seq using 10 ng of input DNA.
Looking first at the sequencing libraries themselves, the 100 pg and 25 pg libraries showed "moderate variations." Sequencing demonstrated high correlation between the three libraries and the standard ChIP-seq method.
Xu told IS that the key to enabling such low starting material was that they were able to "reduce the number of purification steps that cause DNA loss." The TELP protocol only has one optional column-purification step and one magnetic bead binding step, while standard ChIP-seq has three column-purification steps and one agarose purification step.
Next, they compared TELP RNA-seq to standard RNA-seq and found that the two were highly correlated. Looking at genes that were upregulated two-fold in mature adipocytes compared to preadipocytes, they identified 886 genes from standard RNA-seq and 869 genes from TELP RNA-seq — 716, or 80.8 percent, of which were common between both methods.
Another advantage of the TELP RNA-seq versus standard RNA-seq is that it preserves strand specificity. In many RNA-seq protocols, ssDNA is usually converted to dsDNA before adding adapter sequences, Xu explained. But with TELP, dsDNA is first denatured into ssDNA and "one adapter sequence is attached to each ssDNA strand before converting them to dsDNA and performing ligation of the second adapter sequence."
While the protocol has advantages in that it can use little input material and preserve strand specificity, Xu said it still has some limitations. TELP does not work well with longer DNA fragments, he said. Its sweet spot is fragments between 100 bp and 800 bp. And while the protocol can work with paired-end reads, "special data processing is needed" due to the homopolymer stretches.
Nonetheless, he said the protocol uses common enzymes and reagents, so is not more expensive. In addition, it is efficient, combining the DNA amplification and library construction together, while "some other methods "need to perform a pre-amplification step for the DNA sample and then construct the sequencing library," Xu said.
Xu said that the group is interested in the possibility of commercializing TELP, but did not elaborate. The researchers are continuing to improve the method, trying to make it even simpler, more robust, and more sensitive. Currently, he said his lab is testing it in small scale ChIP-seq and RNA-seq and suggested that it could be a good method for analyzing limited clinical samples. In addition, he said he is testing TELP in NGS applications other than RNA-seq and ChIP-seq.