UCSFStanford Oligo Array Meeting

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UCSF/StanfordOligo Array Meeting

• Ash Alizadeh, Max Diehn, Chris Seidel, Joseph
  DeRisi, Mike Hagen, David Erle, Kate Rubins,
  Stephen Popper, Nicki Chin, Joseph Marquis, Elena
  Seraia, John Coller, Jon Pollack, Young Kim, Mike
  Fero, Jean Yang, Andrea Barczak, Peng Zhang, Jing
  Zhu
• April 22, 2004

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Background

• Open questions
• How 3 biased should the oligos be (how biased
  are the various types of labeling reactions)?
• What are the optimal conditions for oligo arrays
  to allow us to answer the above
• i. Printing issues (substrates, amino
  modification, concentration, density)
• ii. Post-processing (substrates, shampooing,
  boiling, BSA, alkylation)
• iii. Labeling (direct vs indirect Cy
  incorporation, amplification, differential
  expression)
• iv. Hybridization (stringency, formamide vs
  SSC/SDS, temperature)
• v. Post-hyb washing (stringency)

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Oligo distances Operon mouse v2 set(David Erle
UCSF)
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Qiagen/Operon
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The Problem
dT primed, 3 biased amp method
Rosetta Method
Genome Biology 2003, 4R66 (http//genomebiology.c
om/2003/4/10/R66)
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Affymetrix On Tiling Controls-I

• What 3'/5' ratio for control genes, for example
  GAPDH and Actin, should I anticipate to obtain on
  GeneChip probe arrays?
• In addition to the conventional probe sets
  designed to be within the most 3' 600 bp of a
  transcript, additional probe sets in the 5'
  region and middle portion (M) of the transcript
  have also been selected for certain housekeeping
  genes, including GAPDH and Actin. Signal
  intensity ratio of the 3' probe set over the 5'
  probe set is often referred to as the 3'/5'
  ratio. This ratio gives an indication of the
  integrity of your starting RNA, efficiency of
  first strand cDNA synthesis, and/or in vitro
  transcription of cRNA. The signal of each probe
  set reflects the sequence of the probes and their
  hybridization properties. A 11 molar ratio of
  the 3' to 5' transcript regions will not
  necessarily give a signal ratio of 1.

http//www.affymetrix.com/support/help/faqs/ge_ass
ays/faq_17.jsp
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Affymetrix On Tiling Controls-II

• What 3'/5' ratio for control genes, for example
  GAPDH and Actin, should I anticipate to obtain on
  GeneChip probe arrays?
• There is no single threshold cutoff to assess
  sample quality for all of the diverse organisms
  and tissues. This is due to the presence of
  different isoforms of these house-keeping genes
  and their different expression patterns in
  various tissues and organisms. Although we
  routinely refer to a threshold ratio of less than
  3 for the most common tissues, such as mammalian
  liver and brain, this may not be applicable to
  all situations. It may be more appropriate to
  document the 3'/5' ratios within a particular
  study and flag the results that deviate,
  therefore representing an unusual sample that
  deserves further investigation.

http//www.affymetrix.com/support/help/faqs/ge_ass
ays/faq_17.jsp
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RNA digestion plot shows strong dependency on
chip design identical biological probes
HG_U95Av2
HG_U133A 10/11 probes from U95Av2 used
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Oligo distances Operon mouse v2 set(David Erle
UCSF)
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Distance from 3 end vs log2 spot
intensity(David Erle, UCSF)
purple aRNA blue cDNA yellow pred aRNA lt green
aRNA- pred aRNA
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Is comparing 70mers at various distances from
different genes fair?
Average Log(2) Unigene Cluster Size (mRNAsESTs)
Representative mRNA size per UG Cluster
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Our tiling experiment

• Tiled 16 long mouse mRNAs, known to be expressed
  at high levels in lung by Operon arrays and
  Affymetrix arrays (picked by David Erle)
• Best 70mer picked for each exon (Ash
  Alizadeh/Chris Seidel)
• Added 10 housekeeping mouse mRNAs supplemented
  without attention to exon structure
• 3 Random sequences, 4 empty wells

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Microarray Versions

• Tiling 1 3 plates printed on PLL, Schott AS,
  Corning UltraGAPS, 6 spots per well
• Mm tiling plate, 300 uM (accidental)
• Hs tester plate from Illumina (unmodified 70mers
  at 50, 40, and 20 uM)
• Hs tester plate from Qiagen/Operon (c-6 amino
  modified 69mers at 50, 40, and 20 uM)
• Tiling 2 9 plates printed on PLL, Schott AS,
  Corning UltraGAPS, 6 spots per well (3 tandem
  replicates, each plate printed 2x)
• Largely same as tiling 1, added Version 2 of
  amino-mod from Illumina for tiling plate and MJDC
• corrected 300 uM Mm tiling to 50 and 25 uM, did
  this for both amino mod versions (V1 and V2)
• Also printed 192 MJ 70mers at 25 and 50 uM, V1
  and V2
• Tiling 3 (in progress)
• Increased density, sharper tips
• No human spots
• 4 spot replicates per print-plate well
• Titrated down to 2uM (2-50 uM)

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Mike Hagen Ti1n014 Hagen heart vs lung prep 1
(rescanned 2 days later to be subsat)
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Mike HagenTi2n014? Hagen heart vs lung prep 2
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Kate RubinsTi2n039 Falkow Spleen Total vs.
Stratagene Reference
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Kate Rubins Ti2n040 Stratagene Total RNA Spleen
vs. Stratagene Total RNA Brain
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Nicki Chin Ti2n095 Stratagene Ref vs Stratagene
Ref PLL 42C, 30 formamide, 5XSSC
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Nicki Chin Ti2n073 Stratagene Ref vs Stratagene
Ref AS 42C, 30 formamide, 5XSSC
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Part II-Oligo Array Conditions

• Oligos produce bigger spots with older SFGF tips
  (120 uM median spot size) spotting size seems
  independent of substrate and printing
  concentration
• cDNA protocols do NOT directly apply to oligo
  arrays
• Boiling after SA/NMP causes catastrophic failure
• Not shampooing causes faint glow to many spots,
  which appears non-specific
• Oligos printed on cDNA arrays seem to confound
  cDNA measurements when arrays treated w/
  conditions optimized for cDNAs (Kate Rubins LC
  smallpox ORF 70mers)
• Suspect oligos cross-contaminating each other
  (DNA jumping) during post-processing without
  shampoo

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Dense Packing
Regular Packing
r
r
x
r
r/2
x2 (r/2)2 r2 x 0.866 r Thus, when you dense
pack, you reduce one dimension by 13.4 at the
expense of increasing the other dimension by
1/(2Nc) where Nc is the number of columns. This
increase in width is neglegible in the limit of
large Nc, so in the limit of large Nr and Nc the
gain in packing is 13.4.
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Effect of printing concentration on intensity (by
vendor) on Ti1 PLL array Sybr Green
(SFGF)random 9mer (UCSF Sandler Core)
Sybr Green Stain
Nonamer Hyb
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5-Amino Modification
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Effect of printing concentration on hybridization
intensity (Nonamers on AS Ti2 array)
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Effect of Illuminas V1 vs V2 amino modification
on real hybridization Ti2010 (Mike Hagen)
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Effect of Illuminas V1 vs V2 amino modification
on real hybridization Ti2010 (PLL, Mike Hagen)
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Cy3 and Cy5 average intensity values for 96
oligos printed in duplicate at the concentrations
indicated. Median background for Cy3 and Cy5 was
71 and 47 respectively. So I printed Operon at 4
diff concentrations, and illumina at 2 diff
concentrations (several replicates of each spot),
and just took the averages of each group.
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Effect of printing concentration on random 9-mer
signal on PLL (left) and AS (right) using Tiling
3 array (Andrea Barczak, UCSF)
PLL_9mer.0116a.gpr
Schott_AS_9mer.0026.gpr
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Post-Processing
BSA instead of alkylation is commonly preferred
for AS, and some PLL users use BSA too, though no
analysis has been published
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BSA vs Anhydride Blocking (SFGF MM cDNA QC
arrays)
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42 deg (30 formamide Corning Protocol) vs 65
deg (Ash/Max protocol) hyb (Nicki Chin, SFGF MM
QC cDNA arrays)
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Array CGH of female vs male genomic DNA (Jon
Pollack Young Kim)UCSF Protocol From Andrea
Barczak
T test UCSF vs sheg 8.77 x 10-8
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UCSF Good v Bad (-35 cutoff) T test 0.147
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pick70_oligos ---------------------------------
-------------------------- Field
Type Null Key Default Extra
-------------------------------------------
---------------- oligo_name
varchar(25) PRI
pick70_name varchar(120)
sequence varchar(80)
percent_gc
float YES NULL
int_repeat smallint(6) YES
NULL self_anneal
smallint(6) YES NULL
pick70_target_id varchar(120) YES
NULL energy float
YES NULL
-------------------------------------------
----------------
pick70_secondary -------------------------------
---------------------------- Field
Type Null Key Default Extra
-------------------------------------------
---------------- oligo_name
varchar(20) MUL
secondary_target varchar(20)
position int(11)
YES NULL energy
float 0
sec_target_seq varchar(150) YES
NULL ----------------------------
-------------------------------
oligo2target ---------------------------------
------------------- Field Type
Null Key Default Extra ---------------
-------------------------------------
oligo_name varchar(25) PRI
target_id varchar(20) MUL
id_source varchar(20)
YES NULL -----------------
-----------------------------------
mousdb_islands ---------------------------------
------------------------------ Field
Type Null Key Default
Extra ---------------------------------------
------------------------ c_id_ex_i
varchar(20) PRI
c_id_ex varchar(15)
MUL c_id
varchar(15) MUL
chr varchar(5) YES
NULL strand char(1)
YES NULL
left_bound int(10) unsigned YES
NULL right_bound int(10)
unsigned YES NULL
5_marg mediumint(8) unsigned YES
NULL 3_marg mediumint(8)
unsigned YES NULL
freq_full smallint(5) unsigned YES
NULL full smallint(5)
unsigned YES NULL
freq_all_t smallint(5) unsigned YES
NULL all_t smallint(5)
unsigned YES NULL
n_min smallint(5) unsigned YES
NULL n_max smallint(5)
unsigned YES NULL
defline varchar(120) YES
NULL sequence text
YES NULL
ex_type varchar(5) YES
NULL refseq tinyint(1)
YES NULL
--------------------------------------------
-------------------
mousdb_exons-----------------------------------
---------------------------- Field
Type Null Key Default
Extra ---------------------------------------
------------------------ c_id_ex
varchar(15) PRI
c_id varchar(15)
MUL chr
varchar(5) YES NULL
strand char(1) YES
NULL left_bound int(10)
unsigned YES NULL
right_bound int(10) unsigned YES
NULL 5_marg mediumint(8)
unsigned YES NULL
3_marg mediumint(8) unsigned YES
NULL freq_full smallint(5)
unsigned YES NULL full
smallint(5) unsigned YES
NULL freq_all_t smallint(5)
unsigned YES NULL
all_t smallint(5) unsigned YES
NULL defline varchar(120)
YES NULL
sequence text YES
NULL ex_type varchar(5)
YES NULL refseq
tinyint(1) YES NULL
------------------------------------
---------------------------
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Splicing examples

• Hs SORBS1 hg16dev chr10.838 -gt 12 skips

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Rules for picking a 70mer from exons for a gene

• 70mer penalty score ( of 2ndary hits)
  (sum of 32ndary
  energies/-30)
  3(distance from 3' end)/1500
  evidence score
• where evidence score 20 - 20
  (eT-e.min/e.max-e.min)
• eT is the denominator in the last field within
  the defline and represents the total evidence
  available for that exon,
• e.min represents the minimum denominator seen
  among the exons, and
• e.max represents the max denominator seen among
  the exons.
• The 3 in the second and third terms is an
  arbitrary constant, and 20 in the 3rd term is
  just an arbitrary constant. The idea is to give
  20 points to the exon with the least amount of
  evidence and give 0 points to the exon with the
  most evidence.
• After the scores are calculated for each exon,
  we pick the one with the lowest penalty score.

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What is the average ratio of transcripts for a
given exon relative to the exon with maximal
evidence, starting from the last exon (3 most
exon is farthest left)
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dT vs pdN6 on total RNARDP108 vs 110 (MH)
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cDNA/direct vs. aRNA/cRNA/AARevisiting GAPD
Jess Leber Exp1, 050404, rdb097 cDNA (top) vs
rdb098 cRNA (bottom) Brain (Cy3) vs Testis
(Cy5) No fragmentation all blocking reagents in
cDNA, no Cot-1 DNA in cRNA centricons at all
steps for both
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cDNA/direct vs. aRNA/cRNA/AAExaggerated 3 bias
Jess Leber Exp1, 050404, rdb097 cDNA (top) vs
rdb098 cRNA (bottom) Brain (Cy3) vs Testis
(Cy5) No fragmentation all blocking reagents in
cDNA, no Cot-1 DNA in cRNA centricons at all
steps for both
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cDNA/direct vs. aRNA/cRNA/AADisappearing Genes
Jess Leber Exp1, 050404, rdb097 cDNA (top) vs
rdb098 cRNA (bottom) Brain (Cy3) vs Testis
(Cy5) No fragmentation all blocking reagents in
cDNA, no Cot-1 DNA in cRNA centricons at all
steps for both
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Optimal Printing Concentration on PLL and ASTop,
PLLRDP110, Mike Hagen Bottom, ASTP3023 Andrea
Barczak)
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't Hoen PA et alNucleic Acids Res. 2003 Mar
131(5)e20 http//nar.oupjournals.org/cgi/conten
t/full/31/5/e20
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't Hoen PA et alNucleic Acids Res. 2003 Mar
131(5)e20 http//nar.oupjournals.org/cgi/conten
t/full/31/5/e20
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Amp vs Unamp

• JL's first set of hybs rdb97 vs 98 shows
  accelerated decay for genes in amplified cRNA
  specimens (scl46610 and scl26041) complete loss
  of signal for many tiled genes with cRNA
  (scl24400 and scl34820) this decay phenomenon
  does NOT improve by fragmenting cRNA (rdb100).
  leaving out all the blocking reagents does not
  rescue loss of signal in cRNA. JL's cDNA decay
  kinetics seem less worrisome than most direct inc
  cDNA hybs we've seen before, where the signal
  decay was as bad as 90 drop in 1kb. why is
  this? we suspect RNA quality, since the brain RNA
  JL used in cy3 was the same as that used by MH
  for rdp108, where the decay seems comparable.
  accordingly, the best RNAs now show lt2-fold drop
  in 1kb for cDNA using direct cy-dutp

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