S. pombe 70-mer Genome Set
Malcolm Cook, Madelaine Marchin, and Chris Seidel
Stowers Institute for Medical Research, Kansas City, Missouri, USA
Comparison: Affy vs. Spotted Array
Melting Temperature
We have designed a publicly available 70-mer probe set for producing DNA
microarrays to measure gene expression in S. pombe. The probe set includes
probes for exons, introns, and 3’ UTRs. The set will make investigations into
gene expression more accessible to the community.
2000
Melting Temperature for All Probes
Frequency
Number of Probes in Set
8785
6918
Introns
1521
Mitochondrial
13
65
70
75
80
Melting Temperature (Degrees Celsius)
Design Process
Melting temperature refers to the temperature at which half the molecules
between the oligo and sample are bound and half are not. As the
temperature increases the likelihood of dissociation increases.
3’ UTRs
Find 3’ UTRs longer than
70. Get their sequences
and add to 3’ UTR file in
fasta format.
0.30
Introns
Find introns longer than
70. Get their sequences
and add to intron file in
fasta format.
Histogram of Probe Intensity
Melting temperature depends on oligo composition. C-G pairs have 3
hydrogen bonds, A-T pairs have 2.
We calculated melting temperature for this histogram as follows:
§
Count G Count C
Tm 81.5 - 16.6 ¨¨ 41 length(seq uence)
©
·
500
¸¸
length(seq uence) ¹
-
Take 1000 bases (or all
coding sequence, if < 1000)
from the 3’ end of gene. Add
that sequence to exon file in
fasta format.
B
Distance from 3' end of Gene for Exon Probes
0.00
400
Frequency
600
80
mean: 1052
min:79
max:9404
median:845
0
5
10
15
40
60
Frequency
Sequences in fasta format files
mean: 490
min:69
max:1600
median:445
800
100
120
Distance from 3' end of Gene for Intron Probes
0.05
A
0.10
Distance from 3’ end of Gene
No
Get each exon
(or fractional
exon) sequence,
add to exon file
in fasta format.
Density
Multiple
exons?
Yes
exon
intron
3UTR
0.25
Exons
Find exons longer than
80 and within 1000 bases
(coding sequence) of 3’
end of gene.2
Intensity of Exons, Introns, and 3’ UTRs
0.20
Download Genome
Get chromosome contig
files from Sanger.1
The same experiment (pot1 knockout vs. wild type) was performed using both
Affymetrix arrays and our spotted 70-mer oligonucleotide arrays. We found a
correlation of .809 between our expression ratio of wild type to pot1 knockout
and the ratio of the wild type affy array to the pot1 knockout affy array.
0.15
Exons
500
333
0
3’ UTRs
mean:70.8
min:63.6
max:82.4
median:70.6
1500
S. pombe microarrays that use PCR amplified probes (180-500 bp) have
previously been created [1]. Because our probes are shorter, our arrays have
higher resolution. They can be more precise in determining expression and are
capable of measuring intron retention.
1000
Introduction
20
200
log2(sqrt(R*G))
0
0
AOS
Array oligo
selector3
Intensity is shown as compared between the exon, intron, and 3’ UTR probes.
The intron probes (as expected) show a lower intensity than the exon probes.
0
2000
4000
6000
8000
Distance (bp)
0
500
1000
1500
Distance (bp)
Distance from the 3’ end of the gene was a concern due to truncated cDNAs
that occur when reverse transcriptase does not complete synthesis.
A. The distance of the intron probe starting coordinate from the 3’ end of the
containing gene.
B. The distance of the exon probe starting coordinate from the 3’ end of the
containing gene.
Send 70-mer oligo sequences in fasta format files for
Oligo Synthesis (external company, Invitrogen)
Receive 70-mer oligos in plates, print
oligonucleotide probes on glass slides
and do microarray experiments
Microarray Experiment
1. Chromosome files were downloaded from Sanger (ftp.sanger.ac.uk/pub/yeast/
pombe/Chromosome_contigs/) on December 6, 2003. The files were in extended Embl
(artemis) format and we used BioPerl to parse them.
2. The 1000 base 3’ bias is to compensate for the truncated cDNAs that occur when
reverse transcriptase does not complete synthesis.
3. ArrayOligoSelector (AOS) is freely available open source software
(http://arrayoligosel.sourceforge.net) designed by Zhu, Bozdech, and DeRisi [2] to
select oligos from a given sequence based on the following criteria:
• Uniqueness in genome
• Sequence complexity
• Self binding
• GC content
Genome Browser Views of Probe Set
spom_chr1
3222k
3223k
3224k
Curated Genes (CDS)
SPAC6G10.04c
SPAC6G10.05c
pre-AOS
AU007542.3221955
SPAC6G10.04c.3222521
SPAC6G10.04c.3222520
SPAC6G10.05c.3223682
SPAC6G10.04c.3222798
SPAC6G10.05c.3224469
SPAC6G10.04c.3222979
70-mer
AU007542.3221761
SPAC6G10.04c.3222536
SPAC6G10.04c.3222074
SPAC6G10.05c.3223591
SPAC6G10.04c.3222717
SPAC6G10.05c.3224068
SPAC6G10.04c.3222871
3333k
3334k
3335k
3336k
3337k
3338k
3339k
Curated Genes (CDS)
SPBC17D1.02
SPBC17D1.03c
SPBC17D1.04
SPBC17D1.03c.3333985
SPBC17D1.04.3336155
SPBC17D1.05
pre-AOS
SPBC17D1.02.3331778
SPBC17D1.02.3332767
SPBC17D1.05.3338016
SPBC17D1.03c.3333986
SPBC17D1.02.3333296
Accessibility of Microarrays
While commercial arrays remain economically out of reach for many researchers,
advances in oligo synthesis technology have driven down oligo production
costs to such an extent that custom genome sets are within reach of individual
labs, or a consortium of labs, who choose to produce their own microarrays
using common methods (http://www.microarrays.org). Oligo 70-mers are a well
established reagent for producing microarrays [4]. This oligo set represents
an open design for measuring gene expression in S. pombe, including
measurement of individual introns and exons, and a first step towards a whole
genome tiling array for ChIP chip analysis.
Summary
An experiment was set up using
two samples: wild type S. pombe
was compared with a pot1
(protection of telomeres) knockout.
The corresponding protein,
Pot1, regulates telomeres and
its absence in S. pombe causes
rapid loss of telomeric DNA and
chromosome circularization [3].
One of the microarrays is shown
below. Red spots indicate increased
expression in the pot1 knockout.
Green spots indicate decreased
expression in the pot1 knockout.
spom_chr2
3332k
We also examined the difference in expression ratios between the 3’ UTR and
nearest exon and found they had a correlation of .73, indicating they were
similarly expressed.
A High Speed Linear Servo Arrayer was used to print oligos onto Poly-Llysine coated slides. 261 arrays were printed in a single print run. Oligos
were printed at roughly 40 µM in 3 x SSC buffer. After printing, slides were
blocked with succinic anhydride.
SPBC17D1.03c.3334427
-We designed a publicly available 70-mer probe
set for S. pombe and printed it on microarrays.
-We performed an experiment comparing S.
pombe wild type to a pot1 knockout.
-Our results correlated well (> .8) with similar
results from Affymetrix arrays.
Contact Information
Chris Seidel, Managing Director of Microarray, Stowers Institute
cws@stowers-institute.org
Probe design available to download from
http://research.stowers-institute.org/microarray
Acknowledgements
Thanks to Karin Zueckert-Gaudenz and Brian Fleharty for their work printing,
hybridizing, and analyzing the microarrays.
SPBC17D1.03c.3334428
70-mer
SPBC17D1.02.3331778SPBC17D1.02.3332901
SPBC17D1.02.3332564
SPBC17D1.03c.3334275
SPBC17D1.04.3336083
SPBC17D1.05.3337886
SPBC17D1.03c.3334002
SPBC17D1.03c.3333575
SPBC17D1.03c.3334435
Curated genes from the Duke fungal genome browser for S. pombe
(http://fungal.genome.duke.edu/cgi-bin/spom/) are shown in green. The preAOS sequences we selected are shown in pink. The final oligonucleotide
probes as selected by AOS are shown in red. Introns were highlighted in light
blue and a 3’ UTR is highlighted in yellow.
S. pombe poly(A)+ RNA (pot1- and wt) was converted into amino allyl
labeled cDNA by reverse transcription and subsequently coupled with Cy
dyes. The slide shown was hybridized with a mixture of 70 pmol Cy3 (wt)
and Cy5 (pot1-) labeled probes.
Hybridizations were performed at 63 °C overnight under standard conditions,
and slides were washed at room temperature successively with 0.6X
SSC/0.03% SDS and then 0.06X SSC prior to scanning (see also http://
microarrays.org). Microarray images were acquired with a GenePix 4000B
scanner (Axon Instruments, Foster City, CA). For image analysis, GenePix
pro 6.0 software was used (Axon Instruments).
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