Methods for removing unexpressed probes of Affymetrix Human Genome U219 Array
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Yang Shi • 0
@ea61ff7a
Last seen 4 days ago
Zheng Zhou

Dear community, Is there any methods to remove unexpressed probes of Affymetrix U219? It can not be calling detection p value by paCalls because of the absence of mismatch probes. Thanks in advance!

affyRaw <- oligo::read.celfiles(celFiles)
eset <- oligo::rma(object = affyRaw)
oligo::paCalls(object = affyRaw) #Getting probe level data... Error: no such table: mmfeature
pd.hg.u219 Microarray oligo AffymetrixChip • 298 views
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@james-w-macdonald-5106
Last seen 2 days ago
United States

There are some QC probes that you might use.

> library(pd.hg.u219)
> library(DBI)
> con <- db(pd.hg.u219)
> dbListTables(con)
[1] "featureSet" "pmfeature"  "table_info"

> dbGetQuery(con, "select * from featureSet where man_fsetid LIKE 'AFFX%';")
   fsetid strand                  man_fsetid
1       1      1              AFFX-DapX-5_at
2       2      1              AFFX-DapX-M_at
3       3      1              AFFX-DapX-3_at
4       4      1              AFFX-LysX-5_at
5       5      1              AFFX-LysX-M_at
6       6      1              AFFX-LysX-3_at
7       7      1              AFFX-PheX-5_at
8       8      1              AFFX-PheX-M_at
9       9      1              AFFX-PheX-3_at
10     10      1              AFFX-ThrX-5_at
11     11      1              AFFX-ThrX-M_at
12     12      1              AFFX-ThrX-3_at
13     13      1             AFFX-TrpnX-5_at
14     14      1             AFFX-TrpnX-M_at
15     15      1             AFFX-TrpnX-3_at
16     16      1        AFFX-r2-Ec-bioB-5_at
17     17      1        AFFX-r2-Ec-bioB-M_at
18     18      1        AFFX-r2-Ec-bioB-3_at
19     19      1        AFFX-r2-Ec-bioC-5_at
20     20      1        AFFX-r2-Ec-bioC-3_at
21     21      1        AFFX-r2-Ec-bioD-5_at
22     22      1        AFFX-r2-Ec-bioD-3_at
23     23      1         AFFX-r2-P1-cre-5_at
24     24      1         AFFX-r2-P1-cre-3_at
25     25      1         AFFX-r2-Bs-dap-5_at
26     26      1         AFFX-r2-Bs-dap-M_at
27     27      1         AFFX-r2-Bs-dap-3_at
28     28      1         AFFX-r2-Bs-lys-5_at
29     29      1         AFFX-r2-Bs-lys-M_at
30     30      1         AFFX-r2-Bs-lys-3_at
31     31      1         AFFX-r2-Bs-phe-5_at
32     32      1         AFFX-r2-Bs-phe-M_at
33     33      1         AFFX-r2-Bs-phe-3_at
34     34      1       AFFX-r2-Bs-thr-5_s_at
35     35      1       AFFX-r2-Bs-thr-M_s_at
36     36      1       AFFX-r2-Bs-thr-3_s_at
37     37      1  AFFX-HUMISGF3A/M97935_5_at
38     38      1 AFFX-HUMISGF3A/M97935_MA_at
39     39      1 AFFX-HUMISGF3A/M97935_MB_at
40     40      1  AFFX-HUMISGF3A/M97935_3_at
41     41      1     AFFX-HUMRGE/M10098_5_at
42     42      1     AFFX-HUMRGE/M10098_M_at
43     43      1     AFFX-HUMRGE/M10098_3_at
44     44      1   AFFX-HUMGAPDH/M33197_5_at
45     45      1   AFFX-HUMGAPDH/M33197_M_at
46     46      1   AFFX-HUMGAPDH/M33197_3_at
47     47      1     AFFX-HSAC07/X00351_5_at
48     48      1     AFFX-HSAC07/X00351_M_at
49     49      1     AFFX-HSAC07/X00351_3_at
50     50      1            AFFX-M27830_5_at
51     51      1            AFFX-M27830_M_at
52     52      1            AFFX-M27830_3_at
53     53      1             AFFX-hum_alu_at
54     54      1             AFFX-r2-TagA_at
55     55      1             AFFX-r2-TagB_at
56     56      1             AFFX-r2-TagC_at
57     57      1             AFFX-r2-TagD_at
58     58      1             AFFX-r2-TagE_at
59     59      1             AFFX-r2-TagF_at
60     60      1             AFFX-r2-TagG_at
61     61      1             AFFX-r2-TagH_at
62     62      1           AFFX-r2-TagJ-3_at
63     63      1           AFFX-r2-TagJ-5_at
64     64      1           AFFX-r2-TagO-3_at
65     65      1           AFFX-r2-TagO-5_at
66     66      1           AFFX-r2-TagQ-3_at
67     67      1           AFFX-r2-TagQ-5_at
68     68      1          AFFX-r2-TagIN-3_at
69     69      1          AFFX-r2-TagIN-M_at
70     70      1          AFFX-r2-TagIN-5_at
71     71      1    AFFX-Nonspecific-GC03_at
72     72      1    AFFX-Nonspecific-GC04_at
73     73      1    AFFX-Nonspecific-GC05_at
74     74      1    AFFX-Nonspecific-GC06_at
75     75      1    AFFX-Nonspecific-GC07_at
76     76      1    AFFX-Nonspecific-GC08_at
77     77      1    AFFX-Nonspecific-GC09_at
78     78      1    AFFX-Nonspecific-GC10_at
79     79      1    AFFX-Nonspecific-GC11_at
80     80      1    AFFX-Nonspecific-GC12_at
81     81      1    AFFX-Nonspecific-GC13_at
82     82      1    AFFX-Nonspecific-GC14_at
83     83      1    AFFX-Nonspecific-GC15_at
84     84      1    AFFX-Nonspecific-GC16_at
85     85      1    AFFX-Nonspecific-GC17_at
86     86      1    AFFX-Nonspecific-GC18_at
87     87      1    AFFX-Nonspecific-GC19_at
88     88      1    AFFX-Nonspecific-GC20_at
89     89      1    AFFX-Nonspecific-GC21_at
90     90      1    AFFX-Nonspecific-GC22_at
91     91      1    AFFX-Nonspecific-GC23_at
92     92      1    AFFX-Nonspecific-GC24_at
93     93      1    AFFX-Nonspecific-GC25_at

Those last ones with the Nonspecific in the name are probes that aren't meant to bind to anything but have increasing GC content. At a certain point the GC content gets high enough that the probes will bind to lots of things, so they aren't all useful. You can decide which ones you might want to use by doing something like

> boxplot(t(exprs(eset)[grep("Nonspecific", row.names(eset)),]), xaxt = "n")
> axis(1, at = 1:23, gsub("AFFX-Nonspecific-", "", grep("Nonspecific", row.names(eset), value = TRUE)), las = 2)

I would argue that up to maybe 13 or 14 is useful as a measure of background binding, and you could just remove any probes where a certain proportion of the probesets are below that level.

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Thanks for your excellent reply sir! But there are still some points confused me. (1).dbGetQuery get 93 kinds of probes. Would be okay for just selected those "Nonspecific" for backgroud binding intensity? (2).As your suggested, 13 or 14 maybe used as background binding. What is the criteria? Just based on expression value, like less than 5? Here is the plot drawn by you code. Thanks so much sir. enter image description here

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It's a non-scientific selection. The Nonspecific probes are meant to be sequences that are not found in nature, so any binding is due to cross-hybridization. This plot is one of the reasons that GCRMA was developed, as it's obvious that as the GC content goes up, the non-specific binding goes up as well.

But for your goal, you want to exclude probesets that you think are not really measuring anything, right? And these probes are not supposed to be measuring anything, but there is the added complexity that the high GC probes start to bind due to the GC issue. We need to define some expression level cutoff that represents background binding, without representing background binding that you expect when the GC content gets too high, so you have to decide at which point that starts to happen. I would argue 12 - 15, but it's your analysis, so you should decide for yourself.

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Thanks sir! As your mentioned here, 12-15 means cutoff value about 4? Could I select the reflection point as the "point" that starts to happen?

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We have progressed past the point where I show you how to do things using Bioconductor packages, and into the realm where I start telling you how to do your analysis, which is something I try really hard to avoid. As I mentioned in my last post, this is your analysis, and you want to exclude things, and I have simply showed you the probes that are arguably useful for doing that sort of thing.

What cutoffs you want to use are up to you to decide, because you are the one who will ultimately have to defend your choices.

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Thanks sir! I got it now!

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Guido Hooiveld ★ 3.3k
@guido-hooiveld-2020
Last seen 38 minutes ago
Wageningen University, Wageningen, the …

To add to the extensive discussion above, you may also want to have a look at the so-called UPC algorithm, available in the package SCAN.UPC. "The Universal exPression Codes (UPC) method is an extension of SCAN that estimates whether a given gene/transcript is active above background levels in a given sample." Paper. Be aware, though, that like in the discussion above, you still have to decide yourselves which cutoff value to apply!

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Thanks sir! The normalization of SCAN not only log2 but also center to zero transformation. But in my opinion, the latter (center to 0) is not suitable for some analyses like differentially expressed genes analysis. Besides, is this correct to do UPC after oligo::rma?

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Regarding the normalization method (SCAN); I have only used RMA for the reasons James explained in one of your other threads (Quantile Normalization after rma or not?).

One could consider using the UPC scores to filter your RMA-normalized dataset, similar like you would do using the expression cutoff value you discussed above, or using Affymetrix's P/A calls (if applicable for the array type). You 'just' reduce your dataset based on some information that is considered to be indicative of the expression of a gene (probeset).

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Got that sir, thanks so much!

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