<div class="preformatted">Hi
I have a question regarding the modelMatrix function in limma.
Which has been covered before however I haven't found an appropriate
answer in the
documentation or the list archives.
Can one use modelMatrix to adjust for unequal numbers of technical
replicates? To take a very simplified example for:
targetsx
Cy3 Cy5
1 WT_sample1 Mutant_sa…
Order by: Relevance
Dear all,
I am currently using DESeq2 package to analyze differential expression of 6 RNAseq samples (each condition has 3 biological replicates = 3 independant inoculations).
One sample is problematic as out of 44,480 genes, 780 are highly expressed in that same...Dear all,
I am currently using DESeq2 package to analyze differential expression of 6 RNAseq samples (each condition has 3 bi…
updated 5.7 years ago • DcL-A
context:** I'm pretty intro-level at bioinformatics. I'm using ATACseq datasets. 4 groups (multiple replicates per group) of the following format:
Group 1 = ConditionA:TreatmentX,
Group 2 = ConditionA:TreatmentY,
Group 3 = ConditionB...OR placebo.0hr)).
**Third,** I don't know how to obtain {3⋃4}. Should I simply consider all replicates of group 3 as though they were more replicates of …
updated 6.1 years ago • corinne_hutfilz
no NAs in the data
> which(is.na(tmp))
integer(0)
# this is my design. shows that I have replicates.
> rbind(head(design),tail(design))
studyGTEx studyTARGET
SRR1068687 1 0
SRR1068788 1 0
SRR1068808 1 0
…
updated 9.4 years ago • komal.rathi
Hi,
I am wondering which test is suitable for my case two condition i.e. C (3 replicates) and T (3 replicates)) and what is basically makes difference between "Wald" and "LRT" test implemented in DESeq2. I tested...check interaction between conditions). But what if i only have two condition like Male (15 or more replicate) and Female (15 replicates or more replicate).\*\*
Then in this case wh…
updated 9.7 years ago • unique379
The data has 5 mutants and 6 time-points. Each of the mutants and each time-point has 3 technical replicates brining up the total number of samples to 90. There are roughly 40,000 genes in Barley that I wish to probe for differential...mla6", "rar3", "wt")
cols=data.frame(genotype=rep(gen, each=3*6), timepoint=rep(rep(time, each=3),5), replication=rep(c(1,2,3),30))
cols$timepoint = as.factor(co…
updated 7.9 years ago • sagnik
<div class="preformatted">Hi,
I have 12 dye-swap design microarrays for 6 samples, which means
each
sample has 2 microarrays with dye swap, and we did the analysis using
R
limma package. When I submit the paper, the database asked for
normalized
data per each sample (biological replicate) for dye-swap experiment.
But I
only can get normalized data for each slide, and I have two slides pe…
updated 16.9 years ago • fei tian
4 .csv files containing gene level (transcripts collapsed) counts coming from infected (2 biological replicates) and non-infected (2 biological replicates) generated via RSEM. I dont have access to raw data files.
Their contents
<div class="preformatted">
I have to run preprocess core for qantile normalization of my data.
Data has protein name in column and peak intensity in row.
I have data of different time point. each time point has three
replicates. I did all protein normalization for now but two time
points are not behaving as expected. So I would like to do quantile
normalization using for the data and this …
updated 12.7 years ago • Guest User
a developmental microarray time course with 18 time points
under
one biological condition but no replicates. I would like to rank genes
in order of non-constancy.
The timecourse package seems well suited to this analysis...However, in
the documentation it says nothing about the no replicate case. In Tai
and Speed(1), it seems that the method still works in this special
case.
Does anyone know…
updated 14.5 years ago • Johannes Meisig
analysis hugene 2.0 st microarrays.
My experimental setup is a control with 8 biological replicates then 3 timepoints with 9 replicates. The time points are months apart so it isn't really considered time series
updated 10.0 years ago • hakimelakhrass
<div class="preformatted">As low as 0.5 for pearson and 0.8 for spearman
But I'm not holding up my data as a shining example
Surely if there is a lot of natural variation in the biological
system, then you're going to get large variation between biological
replicates.
-----Original Message-----
From: Naomi Altman [mailto:naomi@stat.psu.edu]
Sent: 28 February 2004 05:20
To: michael watson (…
updated 21.9 years ago • michael watson IAH-C
Hi there,
I googled the question, but could not find an answer that can solve my question, so I post it here.
Thank you so much in advance!!
I recently received a dataset that has already been sequenced. The idea was: for each batch of cell, transfected with one **control** vector and a bunch of **gene overexpression** vector. So, in batch one, I have **one control vector** and **si…
updated 6.7 years ago • timedreamer
Using DEseq2, the `lfcShrink` function with the `apeglm` method requires a single `coef` value to be specified (i.e. it does not allow a contrast to be specified). [Further explanation in the manual][1].
Therefore, I am having trouble creating a design matrix that represents my comparison of interest. Here is my example code:
cells <- c(rep("A", 6), rep("B", 6))
treat &…
updated 6.9 years ago • toddknutson
each was hybdized
to 2 arrays. All arrays have duplicate sets of probes (so there are 4
technical replicates for each biological samples).
After reading the posts by Jenny, Gordon and others, I'm a bit
confused with what should...animal (or in her case, different offsprings from the
same dam) should not be treated as technical replicates, so I'm not
quite sure about using duplicateCorrelation h…
example below.
---
library(DESeq2)
\# Toy setting with 1000 genes, 3 groups, and 4 replicates per group.
genes = 1000
groups = 3
replicates = 4
\# Generate the count table. This is a terrible simulation in...but all I'm trying to do is check my usage of DESeq2.
lambda = rgamma(genes\*groups\*replicates, 200, 5)
counts = matrix(rpois(genes\*…
I used the following script, which works fine at least until exiqon
version 11, since the 4 replicates are regularly distributed:
# identify differentially expressed genes:
design <- modelMatrix(targets, ref="Control...sep="\t")
But the last version of the exiqon arrays have a different design, the
spacing of the 4 replicates is not regular any more. 2 of the
replicates are located withi…
duplicates), and tried to identify differentially expressed genes (DEGs). For starters, technical replicates are not independent replicates and thus they only had 2 replicates for each age group. To my experience, such low...number of replicates could hardly generate any significant results, especially after controlling for the huge number of genes analyzed...one gene for example, they claim a ve…
Hi there, I am currently doing a project for my masters degree on some pre-existing time course RNASeq data of covid progression in a cell line of caco-2. I have time points at 0h, 1h, 2h, 4h, 7h, 12h, 24h and 48h as well as control data at time points 4h, 12h and 48h, for all timepoints there are 3 replicates for the data. I am just wondering what would be the best way to include the control dat…
updated 2.1 years ago • drewhaydensteele
<div class="preformatted">I'm using the timecourse library to calculate Hotelling's T2
statistics,
and would like to convert these to P values (and, ultimately, perform
FDR).
For a 4 time point, 3 replicates (control and treated each), 54K gene
experiment, I'm struggling with the appropriate variables for
transformation.
This paper:
http://bioinformatics.oxfordjournals.org/cgi/content/…
updated 19.8 years ago • aaron.j.mackey@gsk.com
000 genes x 8 total samples). My samples (n=4) belong to either of two groups (A and B). I have two replicates for each sample. So, my samples are:
Sample1\_A\_replicate1
Sample2\_B\_replicate1
Sample3\_A\_replicate1
Sample4...to do a differential gene expression analysis. In the siggenes/SAM documentation, it doesn't mention replicates. How do I go about specifying the class labels?
S…
updated 9.0 years ago • Brian Smith
I'm trying to understand what that means. What does a "0" p value mean?
There were two biological replicates for each population. The p value for each pairwise comparison (comparing the two populations in each biological...sample/replicate) is not zero but then the combined p value shows up as zero. Why is that? I didn't perform these analyses and haven't ever
updated 7.0 years ago • vibhusahni
Hello,
It might be a silly question, but I am struggling with it all day now. Hopefully someone with bright insight may be of any help.
I have this df which contains a column with ID names. Some ID names occur one time, other two, and some even 20 times. In other words some IDs are in 1 row, others in many rows.
What I would like to make is a new column which numbers the replicate IDs (from 1,…
updated 10.4 years ago • b.nota
the highly expressed
genes.
However, I found genes that had zero counts in series of 4 samples (2
replicates in each of 2 conditions), while their log2fold change was
3.
what is the reason for that? or how can this be prevented...I used the nbinomWaldTest and did not replace outliers with the
trimmed
mean since I have only 2 replicates per condition.
I'd appreciate your help very much,
Noa Heni…
me="" meaning.="" no="" of="" or="" p-value:="" parameters:="" ppde="" ppde(<p)="" raw="" remove="" replicate="" replicates="" results="" set="" size:="" some="" suggestion="" suggestions,?we="" thanks="" that="" the="" to="" two="" value="" value:="" when="" window="" with
updated 13.9 years ago • Li Zhang
DGEList(counts=counts(set), group=x)</pre>
If I know that I have a batch effect between first replicates (Ctl1, Trt1) and second replicates (Clt2, Trt2). Would it make sense to introduce it already in the design matrix and
updated 10.1 years ago • tonja.r
the other condition (Organoid Vs Tissue) in the following dataset.
```r
Sample Patient Replicate Batch Condition
O3_BR1 3 1 run2 Organoid
O3_BR2 3 2 run2 Organoid
O3_BR3 3 3 run2 Organoid
O4_BR1 4 1 run2 Organoid...9 1 run1 Tissue
```
I have paired samples, each of them with biological…
updated 4.6 years ago • S
LIMMA. But here each disease has 2-3
arrays from different patient blood samples. These are not true
replicates, per se but thats all we have. My question is how do we
treat them? Can I consider them as replicates? (I understand LIMMA...will
combine the replicate data.)
here is the design........
1. disease1 patient1
2. disease1 patient2
3. disease1 patient3
4. disease2 patient4
5. disease2
LIMMA. But here each disease has 2-3
arrays from different patient blood samples. These are not true
replicates, per se but thats all we have. My question is how do we
treat them? Can I consider them as replicates? (I understand LIMMA...will
combine the replicate data.)
here is the design........
1. disease1 patient1
2. disease1 patient2
3. disease1 patient3
4. disease2 patient4
5. disease2
Hello,
I have a dataset consisting of 16 pooled libraries sequenced on three lanes (2x125bp, 350bp fragment size, 40M reads per library). I isolated RNA from the same type of tissue across different individuals. There are three levels for one condition ("behavior"), and 4-6 biological replicates per level. I assessed the quality of the data using DESeq2 to calculate sample-to-sample VST distance…
gt;viral strains in a complex experiment.
>
>Factors:
>1) Patient (p3, p4, p5)
>2) "Replicate" (a, b, c)
>3) Viral Titer (continuous integer variable)
>4) Viral Strain (O, F, S)
>
>Although all 3 of the "replicates" per...the same
>way, there are significant differences in the amount of virus
>recovered from each "replicate", a…
updated 14.9 years ago • Naomi Altman
ab
0b ------> a0
The arrow points towards RNA sample labeled with Cy5 and I have two
technical replications for each arrow. Therefore there are 6
experiments * 2
replicates = 12 arrays
Here are my questions:
1. Is my experimental...design suitable for limma analysis?
2. Should I combine the technical replicates before I calculate the
liner
model by lm.series, or just treat the 12 array…
<div class="preformatted">Im trying to find the average A values for replicate chips after using
the lmFit function. I can easily identify the average M values for
each
group of replicates, but I dont see a similar listing for the A
values.
I only appear to be generating one average A value for all replicates.
Does the lmFit function do this, or am I doing something wrong...
thanks
Simon.…
updated 22.1 years ago • Simon Melov
have a RNA seq data which I am trying to identify DEGs. Dataset contains three sets of Untreated - 2 replicates and Treated - 2 replicates. My DEseq2 results shows padj values NA and some p values and padj values as zero. A total
updated 7.0 years ago • aishu.jp
the previous questions, but still not quite sure how to deal with my data. I have 3 replicates each of 14 different genotypes (each RNA batch made on a different day), and I would like to compare them all to wildtype...other. In my summarized experiment each sample is a row. Should I try pooling the three replicates? Also how do I generate the com…
updated 7.7 years ago • rattray56
on 4
different platforms (CodeLink, Affymetrix, Agilent and an inhouse cDNA
array), 2 RNA samples, 3 replicates for each on the one color
microarrays, 6 replicates for the two color arrays. I used limma
with
fdr to adjust the p
meant to
compare two conditions. We have 4 slides of microarrays: all four
considered as biological replicates. However, one slide contains 2
replicate arrays (we named top and bottom). These two come from the
same culture. The
updated 17.0 years ago • Yolande Tra
applied.
I was wondering if this is anything to worry about? I have at least 3 biological replicates per sample, and for some replicates for a specific m/z value the intensity before normalization is actually 0, so
updated 9.0 years ago • djtml
RNA seq data of 5 genotypes each of fast and slow development rate categories with two biological replicates per each genotype. My data looks like this:![My ][1]
Our main question is to study differentially expressed genes between...to study the differential gene expression analysis. I am not sure how to account for the biological replicates of genotypes in the model. I have done the anal…
updated 2.2 years ago • kaurprab
it is expected that nearly all genes will have no change and there is little to no variation across replicates (so near technical replication), and then say < 10 genes with very large fold changes. This scenario could occur...in non-biological samples, for example technical replicates plus DE spike ins. The reason this would cause a problem is that the prior is formed according to a high p…
frustrating. One workaround that came up when consulting my
local statistician: to treat technical replicates and biological
replicates equally. But technical replicates have higher correlation
than biological replicates...I calculated a few correlations by hand):
- technical replicate (same biological sample, same dye,
different
chip): median correlation = 0.55
- biological repli…
treated and untreated. Timepoint 0 is untreated, while 10 and 30 are treated.
- 3 biological replicates per sample
Total = 24 libraries
Here is the design matrix I came up with:
<pre>
genotype timepoint replicate condition...timepoint 10 (i.e. R\_10\_R1, R2 & R3).)
__My first question is:__ do I need to include replicates as a factor? These are just biological…
updated 9.2 years ago • athief
Hi,
I have a experimental setup with 2 different strains, 3 different time points, activation/non-activation and 2 replicates. I've been trying to design the model matrix and write the design formula for some time now, but I still get the "error: inv(): matrix appears to be singular" error.
Here is my experimental design matrix:
<pre>
strain time replicates activation
AS 0 …
updated 9.7 years ago • cdemel
which includes a
>coefficient for each mouse in the linear model to account for
technical
>replication. I am somewhat puzzled because the example in the user's
guide
>returns non-estimable coefficients and hence...and I am not a statistician, I am
struggling in
>finding the correct solution. As we have four replicate spots on our
>arrays I cannot use the "dupli…
updated 20.3 years ago • Gordon Smyth
bams into two different rows on the same column?
My 2 bam files for ChIP(IgG) are actually from 2 replicates.
My purpose of doing things above is to merge replicates - which of course can be done in other ways.
Thank you
Hi there,
I am completely stumped in the analysis of my RNAseq data set using limma. But is there is a possibility to extract the mean expression levels for each sample (averaged among 3 biol. replicates and adjusted for block effects) from the lmFit output or can these be calculate with the help of the estimated coefficients...there is a possibility to extract the mean expression levels for eac…
updated 5.8 years ago • anna
Hi
We have RNA-seq data (three biological replicates in a single condition) and we want to calculate TPM with the sole purpose of ranking genes by expression level...Hi
We have RNA-seq data (three biological replicates in a single condition) and we want to calculate TPM with the sole purpose of ranking genes by expression level (in...of relying on the total reads system?
&…
updated 8.2 years ago • colore
<div class="preformatted">Dear All,
I have been using the Limma library in the R package.
I need to write an output of an analysis into a file
of a text or .csv format. I tried using the "save" and
"dump" functions in R to accomplish the same but
without any success. All I get is a 1kb file with a
link to the results file.
How can I save the results(380000 spots) of an
analysis into a test …
by 10x and would like to compare them against each other. They are all in duplicates (two biological replicates for each TP).
How would one go ahead with duplicated?
Do I need to merge the two biological replicates into one sample
updated 5.4 years ago • Assa Yeroslaviz
The [InTAD paper][1] states `InTAD can be applied to any heterogeneous cohort of samples analyzed by a combination of gene expression and epigenetic profiling techniques and integrates either public or custom information of TAD boundaries.` What I am missing is a statement on its application on non-cohort data. We have a murine cell lines profiled with ATAC-seq and RNA-seq with three to four repl…
updated 6.6 years ago • ATpoint
which are often observational case-control by design
and
the peak data is produced in technical replicates. A method for sample
size determination for planning these studies is given. It
incorporates
routines for adjusting...for the expected heterogeneities and imbalances
in the data and the within-sample replicate correlations.
You may access it through the commands:
> source("http://b…
updated 16.3 years ago • Stephen Nyangoma
to identify the genes that are differentially
expressed. I have two groups (eg., F0 and P6) with two replicates (a
and b)
for each group for the analysis. I was little confused which analysis
to
use, either "Analysis using common...dispersions".
I was not sure as to what value to use for prior.n as I have two
groups with
two replicates each. I would appreciate if you could help me in
deciding the…
updated 14.9 years ago • Sridhara Gupta Kunjeti
of differentially-expressed genes, colleagues ask to see the raw expression data, sometimes for all replicates. We usually show the counts-per-million of the fitted data: eg.
`` class(y)[1]  ...fit <- glmFit(y, design, …) ``
`` cpm(fit$fitted.values) ``
These values are the same for all replicates of …
updated 9.6 years ago • david.hughes
someone explain the organisation of the "read_group_tracking" -
files from Cuffdiff. In the column "replicate" is a number
corresponding
to the BAMs which was input.
But the organisation is odd, as you can see:
replicate
1
0
2
3
4
updated 12.0 years ago • Sindre
the command is "cor <-
duplicateCorrelation(MA,design,ndups=2)", but when I do some technical
replication, how can I use this function? (when it comes to technical
replication, ndups will equal to 1,how to compute correlation...within
array
duplication? And I isolate RNA from bacterial not animal bodys, how to
say
biological replication?).
Finally, how to extract information from the e…
updated 20.5 years ago • ? ?
class="preformatted">Dear all,
I have a question regarding the way to properly design biological
replicate and technical replicates.
In the projet, we have two groups of sample : R (respond to the
treatment), NR (no response).
For...each groups, there is several replicates (3) that come from
different patients (so not strictely biological).
Each patient have two sample : one at day one(d1
updated 14.7 years ago • Guillaume Meurice
QUESTION:
Given that 20 % of variance is no explained , how to integrage ( in R
code ) the factor " replication "
I have tried to add in my original code this part :
the line factor_replication = c(1,2,3,1,2,3,1,2,3,1,2,3)
helmert = model.matrix...the scalar product is no 0 ) . Moreover , the
result "said" that the variance explained by replication is 2 %...
It's very low , too low...…
updated 16.2 years ago • gregory voisin
collecting from the same individuals (plants) for all
timepoints. So if I say we have 3 biological replications per
treatment, it means that on Day 0, I inoculate 3 plants for each
treatment, and these 3 plants are used for all...I gave in previous e-mail
> - I have 6 treatments (including the untreated),
> - 3 biological replicates per treatment per timepoint
> - 5 timepoint…
A,B,C,D,E are five tissue types and D and E are from wild condition only. 1,2 and 3 are biological replicates.
I want to perform:
(i) comparison of differentially expressed genes between all tissue types in wild
(ii) comparison...nbsp;differentially expressed genes between between D and E
How should I setup the design with replicates? Is this correct:
<pre>
…
updated 8.1 years ago • bioinf
and two experimental factors (+/+, +/-, -/+ and -/-). In addition, each group has four biological replicates (the first biological replicate of each group was taken at the same time, the second replicates at another and so...on) and a clear batch effect between replicates.
My design matrix looks like this:
design <- model.matrix(~batch+factor.1+factor.2)
<pre> (Interce…
updated 11.0 years ago • Ekarl2
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