Intergenic 70-mer Genome Tiling set
for Saccharomyces cerevisiae
Madelaine Marchin and Chris Seidel
Stowers Institute for Medical Research, Kansas City, Missouri, USA
Introduction
Probe set comparison with Agilent probes
UCSC Genome Browser Views of Probe Set
We have designed a set of 70-mer oligos for tiling the intergenic regions of the
Saccharomyces cerevisiae genome at an average resolution of 250 nucleotides.
The
set consists of 9,405 sequence optimized oligos chosen such that no intergenic region
greater than 360 bases was left uncovered by a probe.
The oligos were screened for
uniqueness and melting temperature.
chr1:
65000
70000
75000
intergenic (BLAT)
chr3:
3.5 _
80000
100000
95000
100000
intergenic
165000
170000
0.20
250
0.15
200
Density
0.10
150
chr3:
3_
50000
100000
250000
Protein-Coding Genes from Saccharomyces Genome Database
300000
chr3:
3_
1-
155000
160000
BLI_06-23-06
165000
170000
1-
AcH4_dSETvsWT
AcH4_dSETvsWT
0-
0-
-2.5 _
-2.5 _
Protein-Coding Genes from Saccharomyces Genome Database
SGD Genes
Protein-Coding Genes from Saccharomyces Genome Database
SGD Genes
The top two browser tracks represent data fromAgilent
44k Arrays(4), in which an
acetylated histone was mapped by ChromatinThe
IP. bottom browser track represents
data from a parallel experiment using YOGi
our array (data courtesy of Bing Li, Workman Lab).
2
0.05
4
0
0.00
6
65
70
75
80
8
Tm
Histograms of the melting
temperatures of the intergenic probe set and the Operon probe set (which has one
probe per ORF).
9
10
11
12
13
14
15
16
Conclusion
While commercial tiling arrays are becoming more common, they are still cost
prohibitive to many groups. Decreasing oligo production costs have brought synth
of custom genome-sized oligo sets within reach of organized groups choosing to
their own microarrays.
This set has proven to off
er higher resolution data for CGH and
ChIP based studies.The design is publicly available at
http://research.stowers-institute.org/microarray/
.
70-mer probes vs. PCR probes
5000
10000
Summary
15000
20000
uber
25000
30000
35000
uber
0-
AOS
-1.821 _
2.503 _
A comparitive genomic hybridization was performed using the tiling probes. Yeast strains SK1
and S288c were compared. A representation of
the yeast genome is shown.
17
Shown at right is a typical microarray image from a
Chromatin IP experiment using the intergenic tiling set in
conjunction with the Operon Expression The
set. top 7
rows of each grid are the Operon ORF oligos, whereas
the remaining rows consist of intergenic tiling oligos.
Qualitatively, the sets are nearly
indistinguishable.
Array oligo
selector2
150000
200000
BLI_06-23-06
1
100
50
Frequency
0-
SGD Genes
5
chr4:
2.503 _
yogi
Send 70-mer oligo sequences in fasta format files for
Oligo Synthesis (external company, Invitrogen)
-We created a design for an intergenic tiling set for yeast.
-In combination with the commercially available Operon
Expression set, one can produce whole genome microarrays
capable of collecting data at higher resolution than previously
available.
Contact Information
Chris Seidel, Managing Director of Microarray, Stowers Institute
cws@stowers-institute.org
Acknowledgements
yogi
2. ArrayOligoSelector (AOS) is freely available open source software
(http://arrayoligosel.sourceforge.net) designed by Zhu, Bozdech, and DeRisi [1] to
select oligos from a given sequence based on uniqueness in genome, sequence
complexity, self binding, and GC content.
150000
160000
AcH4
CGH with the Tiling array
Array Image
1. SGD_features.tab file was downloaded from SGD (ftp.stanford.edu/pub/yeast/data_
download/chromosomal_feature/) in December 2005. The file was parsed with a perl
script and coordinates of the intergenic regions were determined.
145000
155000
-1 _
Protein-Coding Genes from Saccharomyces Genome Database
SGD Genes
7
Receive 70-mer oligos in plates, print
oligonucleotide probes on glass slides
and do microarray experiments
150000
3
200
400
600
800
1000
Long oligos adhere well to glass, can be bioinformatically optimized for high
city
specifi
Distance between probes (bases)
to a target sequence with low cross hybridization to other sequences, and are not
subject to the synthesis failures commonly encountered with PCR. Initial studies
Histogram of the distances between probes. The
indicated that 70-mers work well as array reagents for ChIP chip based studies, and probes with longer distances between them are reoffers the advantage of generating higher resolution data.
Thus we sought to make
gions where a suitable probe could not be found.
an array consisting entirely of 70-mers to represent the yeast genome to replace the
current PCR product based arrays.
Sequences in fasta format files
145000
1-
-1 _
Protein-Coding Genes from Saccharomyces Genome Database
mean:257, min:116, max:1005, median: 257
Intergenic Regions > 140
Must Tile probes evenly
through region leaving no
region > 360 without a
probe.
chr3:
3.5 _
0-
intergenic (BLAT)
Background
Intergenic Regions 70 - 140
Take entire region. Pick
random strand. Add
sequence to file in fasta
format.
300000
AcH4
105000
Operon
Intergenic
Intergenic Regions < 70
Currently too short to
measure with a 70-mer.
Ignore.
250000
1-
90000
70−mer Tm, Operon ORF versus Intergenic
Download Genome
Get SGD_features.tab
from SGD.1
200000
AcH4
AcH4
chr3:
The set was designed for the production of yeast genome DNA microarrays and was
The probes are shown on the UCSC Genome Browser (http://genome.ucsc.edu).
printed on glass using conventional techniques. While all intergenic regions across
Curated genes and other features from SGD (http://www.yeastgenome.org) are shown
the genome are covered at moderate resolution, the Open Reading Frames (ORFs)
in blue. The intergenic tiling probes are shown in black. Sections shown are from
are not represented, with the exception of chromosome 3 for which both the intergenic
chromosome 1 and chromosome 3, which was tiled at ~250 bp resolution (ORF and
regions and open reading frames are tiled.
intergenic regions).
When used in conjunction with the commercially available expression oligo set
from Operon Biotechnologies, this set
fers
of nearly full genome coverage at higher
Distance between probes
resolution than was previously available, making it ideal for studies involving chromatin
Distance between intergenic tiling probes (S. cerevisiae)
immunoprecipitation (ChIP), and comparative genomic hybridization (CGH).
Design Process
150000
Protein-Coding Genes from Saccharomyces Genome Database
SGD Genes
SGD Genes
Groups printing their own yeast arrays have traditionally relied on PCR products to
represent the genome as ORF and intergenic segments (1,A 2).
successful alternative
to PCR products for construction of expression arrays has been the use of 70-mer
oligonucleotides (3).
50000
intergenic
0-
Thanks to Jennifer Gerton for the 70-mer vs. PCR data and to Bing Li for the Agilent
comparison data.
-1.821 _
Protein-Coding Genes from Saccharomyces Genome Database
SGD Genes
The data presented above represents log base 2 ratios (R/G) from a Chromatin IP
experiment designed to map cohesin along the chromosome.
The top browser track
illustrates data obtained from PCR product arrays
one (probe for each gene and
intergenic region). The bottom track
shows the same experiment using our YOGi
arrays (intergenic tiling set + operon probes, one per ORF). The 70-mers offer higher
resolution data. (data courtesy of Jen Gerton).
References
(1) DeRisi JL, Iyer VR, Brown PO. (1997) Science. 278(5338):680-6.
(2) Reid JL, Iyer VR, Brown PO, Struhl K. (2000) Mol Cell. 6(6):1297-307.
(3) Seidel, C., J.Ten Bosch, J., Batra, S., Lam, H., Tuason, N., Saljoughi, S., and Saul, R. (2000). 70-mer
Oligonucleotides for DNA Microarrays. Abstract. Yeast Genetics and Molecular Biology Meeting, Seattle.
(4) Pokholok DK, Harbison CT, Levine S, Cole M, Hannett NM, Lee
TI, Bell GW, Walker K, Rolfe PA,
Herbolsheimer E, Zeitlinger J, Lewitter F, ford
Gif DK,Young RA. Genome-wide map of nucleosome acetylation
and methylation in yeast. (2005) Cell. 122(4):517-27.