Bisulfite Padlock Probes (BSPP)

Library-free methylation sequencing with bisulfite padlock probes

Dinh Diep1,2,4, Nongluk Plongthongkum1,4, Athurva Gore1,4, Ho-Lim Fung1, Robert Shoemaker3 & Kun Zhang1,2

(Published in Nature Methods)

Protocols

Note: There are two sets of slightly different protocols, one for longer oligonucleotide libraries (>120 mers, such as Agilent’s oligos), and the other for shorter oligonucleotide libraries (<= 100 mers, such as LC Sciences’ oligomix). The reason for creating two different sets of protocols is that shorter oligonucleotidesdo not have enough space to accommodate a number of restriction digestion sites and other sequence feature. Here is a comparison of the two types of oligonucleotide libraries.

  1. Probe preparation:
  • Long oligonucleotides: The technical detail of this probe preparation method was first reported in Zhang et al. Nature Methods, 2009. In this protocol, oligo pools are amplified by one or two rounds of PCR. The double-stranded PCR amplicons are converted into single-stranded DNA by lambdaexonuclease digestion. The 3’-end amplification adaptor sequence is removed by oligo-guided restriction enzyme (Dpn II) digestion on single-stranded DNA. Then 5’-end amplification adaptor sequence is removed by USER enzyme digestion. The advantage of this method is that there is no restriction in the padlock sequences, even the ones containing Dpn II cutting site can be prepared with this protocol. Probes prepared using this protocol have been used in Deng et al. NBT, 2008; Li et al. Science, 2009; Wang et al. GR, 2010; Gore et al. Nature, 2011; Noggle et al. Nature 2011.
  • Short oligonucleotides: The technical detail of this probe preparation method was first reported in Porreca et al, Nature Methods, 2007. It uses two nicking enzymes (Nt.AlwI, Nb,BsrDI) to remove the amplification adaptors. This method has fewer steps and is easier. The limitation is that the padlock probes cannot contain the recognition sites of the two nicking enzymes.
  1. Target capture and sequencing library preparation: due to the difference in the padlock linker sequence between the long and short oligonucleotides, different sets of PCR primers and sequencing primers are used.

AVAILABLE DATA

BSPP probe set:

CGI probe set:

Methylation data in BED format

Note: The reads were mapped to hg18. These files can be visualized using the Human Genome Brower Gateway (http://genome.ucsc.edu/cgi-bin/hgGateway) by loading all the files as custom tracks.
Alternatively, each of the files can be loaded to the Genome Browser using the following web links:

  1. PGP1-Fibroblast
  2. PGP1-iPS Replicate 1
  3. PGP1-iPS Replicate 2
  4. H1 ESC

Raw Illumina sequencing reads

SOFTWARE

bisReadMapper: a pipeline for efficient mapping of targeted and whole genome bisulfite sequencing reads

ppDesigner: software to design padlock probes