Supplementary web site for
Library-free Methylation Sequencing with Bisulfite Padlock Probes
Dinh
Diep*, NongLuk Plongthongkum*, Athurva Gore*, Ho-Lim
Fung, Robert Shoemaker, Kun Zhang.
BSPP probe set.
·
DMR330k
Probe sequences and annotations.
·
LC4k
probe sequences and annotations.
CGI probe set.
·
Note: CGI probes were designed to capture all annotated CpG Islands on Hg19. Two versions of the probes are available, one which is long (~150bp) and should use the experimental protocols designed for long oligonucleotides, and the other which is short (~120bp), and should use the experimental protocols designed for short oligonucleotides.
·
Long CGI
probe sequences and annotations.
·
Short CGI
probe sequences and annotations.
Experimental 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 oligonucleotides do 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 lambda exonuclease 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.
2. 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.
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;
4. H1
ESC.
Raw Illumina
sequencing reads.
bisReadMapper: a pipeline for efficient mapping of targeted and whole
genome bisulfite sequencing reads.
ppDesigner: software to design padlock probes.
Please contact Dr. Kun Zhang (kzhang _at_ bioeng.ucsd.edu) for questions.