<html><body style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space; "><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; color: rgb(179, 17, 0); "><font class="Apple-style-span" color="#00169A"><font class="Apple-style-span" color="#000000">Dear All,</font></font></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; color: rgb(179, 17, 0); "><font class="Apple-style-span" color="#000000">I have being working with pair samples for 3 subjects using edgeR package</font></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; color: rgb(179, 17, 0); "><font class="Apple-style-span" color="#000000">and I am puzzle with the results of my normalization. After normalization, the data is skewed towards the LS group, and as a result, I get much more genes up than down-regulated. We have study this disease extensively in large samples with microarray and this is not the case there, so now I am suspicious of my normalization.</font></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; color: rgb(179, 17, 0); "><font class="Apple-style-span" color="#000000">I am including teh code and a pdf with the smear plot using the normalization options in edgeR. On all of them the data looks worst than after normalization. </font></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; color: rgb(179, 17, 0); "><font class="Apple-style-span" color="#000000">If someone can look to what I did and point to any mistake, I will really appreciate.</font></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; color: rgb(179, 17, 0); "><font class="Apple-style-span" color="#000000">I dont know if the point is that I am deleting the unmapped reads before normalization. </font></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; color: rgb(179, 17, 0); "><font class="Apple-style-span" color="#000000">I was instructed as such in the SeqAnswer forum.</font></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; color: rgb(179, 17, 0); "><font class="Apple-style-span" color="#000000"><br></font></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; color: rgb(179, 17, 0); "><font class="Apple-style-span" color="#000000"><br></font></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; color: rgb(179, 17, 0); "><font class="Apple-style-span" color="#000000">## Reading Files</font></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; color: rgb(179, 17, 0); "><span style="color: #000000">files</span><span style="color: #00169a"> <- </span><span style="color: #000000">dir</span><span style="color: #00169a">(</span><span style="color: #000000">pattern</span><span style="color: #00169a">=</span>"*\\counts.txt$"<span style="color: #00169a">)</span></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; ">files.pheno<span style="color: #00169a"><-</span>data.frame<span style="color: #00169a">(</span>files<span style="color: #00169a">=</span>files<span style="color: #00169a">, </span>group<span style="color: #00169a">=</span>factor<span style="color: #00169a">(</span>substr<span style="color: #00169a">(</span>files<span style="color: #00169a">,</span><span style="color: #005218">1</span><span style="color: #00169a">,</span><span style="color: #005218">2</span><span style="color: #00169a">),</span>levels<span style="color: #00169a">=</span>c<span style="color: #00169a">(</span><span style="color: #b31100">"NL"</span><span style="color: #00169a">,</span><span style="color: #b31100">"LS"</span><span style="color: #00169a">)), </span>Patient<span style="color: #00169a">=</span>factor<span style="color: #00169a">(</span>substr<span style="color: #00169a">(</span>files<span style="color: #00169a">,</span><span style="color: #005218">3</span><span style="color: #00169a">,</span><span style="color: #005218">4</span><span style="color: #00169a">)))</span></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; color: rgb(0, 22, 154); min-height: 19px; "><span class="Apple-style-span" style="color: rgb(0, 0, 0); ">PScounts<span style="color: #00169a"> <-</span>readDGE<span style="color: #00169a">(</span>files.pheno<span style="color: #00169a">)</span></span></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; ">colnames<span style="color: #00169a">(</span>PScounts<span style="color: #00169a">)<-</span>paste<span style="color: #00169a">(</span>PScounts<span style="color: #00169a">$</span>samples<span style="color: #00169a">$</span>group<span style="color: #00169a">,</span>PScounts<span style="color: #00169a">$</span>samples<span style="color: #00169a">$</span>Patient<span style="color: #00169a">,</span>sep<span style="color: #00169a">=</span><span style="color: #b31100">'-'</span><span style="color: #00169a">)</span></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; color: rgb(179, 17, 0); "><font class="Apple-style-span" color="#000000"><br></font></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; color: rgb(179, 17, 0); "><font class="Apple-style-span" color="#000000">##delete unmmaped reads</font></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; color: rgb(179, 17, 0); "><span style="color: #000000">unmmaped</span><span style="color: #00169a"><-</span><span style="color: #000000">c</span><span style="color: #00169a">(</span>'no_feature'<span style="color: #00169a">,</span>'ambiguous'<span style="color: #00169a">,</span>'not aligned'<span style="color: #00169a">,</span>'too low aQual'<span style="color: #00169a">)</span></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; ">PScounts<span style="color: #00169a"><-</span>PScounts<span style="color: #00169a">[-</span>which<span style="color: #00169a">(</span>rownames<span style="color: #00169a">(</span>PScounts<span style="color: #00169a">$</span>counts<span style="color: #00169a">)%</span><span style="color: #c68800">in</span><span style="color: #00169a">%</span>unmmaped<span style="color: #00169a">),]</span></div><div apple-content-edited="true"> <div style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space; "><span class="Apple-style-span" style="border-collapse: separate; color: rgb(0, 0, 0); font-family: Helvetica; font-size: medium; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; orphans: 2; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-border-horizontal-spacing: 0px; -webkit-border-vertical-spacing: 0px; -webkit-text-decorations-in-effect: none; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; "><div style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space; "><span class="Apple-style-span" style="border-collapse: separate; color: rgb(0, 0, 0); font-family: Helvetica; font-size: medium; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; orphans: 2; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-border-horizontal-spacing: 0px; -webkit-border-vertical-spacing: 0px; -webkit-text-decorations-in-effect: none; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; "><div style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space; "><div><div><br></div><div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; "><span class="Apple-style-span" style="color: rgb(80, 79, 79); ">#Calculate Normalizations </span></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; color: rgb(179, 17, 0); "><font class="Apple-style-span" color="#00169A"><font class="Apple-style-span" color="#000000"><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; "><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; ">d.PS<span style="color: #00169a"> <- </span>calcNormFactors<span style="color: #00169a">(</span>PScounts<span style="color: #00169a">)</span></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; color: rgb(179, 17, 0); "><span style="color: #000000">pdf</span><span style="color: #00169a">(</span>'Normalization Plots.pdf'<span style="color: #00169a">,</span><span style="color: #000000">height</span><span style="color: #00169a">=</span><span style="color: #005218">10</span><span style="color: #00169a">,</span><span style="color: #000000">width</span><span style="color: #00169a">=</span><span style="color: #005218">10</span><span style="color: #00169a">)</span></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; ">layout<span style="color: #00169a">(</span>matrix<span style="color: #00169a">(</span><span style="color: #005218">1</span><span style="color: #00169a">:</span><span style="color: #005218">4</span><span style="color: #00169a">,</span><span style="color: #005218">2</span><span style="color: #00169a">,</span><span style="color: #005218">2</span><span style="color: #00169a">,</span>byrow<span style="color: #00169a">=</span><span style="color: #c68800">TRUE</span><span style="color: #00169a">))</span></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; ">a<span style="color: #00169a"><-</span>plotSmear<span style="color: #00169a">(</span>PScounts<span style="color: #00169a">, </span>panel.first<span style="color: #00169a">=</span>grid<span style="color: #00169a">(),</span>smooth.scatter<span style="color: #00169a">=</span><span style="color: #c68800">FALSE</span><span style="color: #00169a">,</span>main<span style="color: #00169a">=</span><span style="color: #b31100">'before normalization'</span><span style="color: #00169a">)</span></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; ">ma.plot<span style="color: #00169a">(</span>a<span style="color: #00169a">$</span>A<span style="color: #00169a">,</span>a<span style="color: #00169a">$</span>M<span style="color: #00169a">,</span>plot.method<span style="color: #00169a">=</span><span style="color: #b31100">'add'</span><span style="color: #00169a">,</span>cex<span style="color: #00169a">=</span><span style="color: #005218">0</span><span style="color: #00169a">)</span></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; ">b<span style="color: #00169a"><-</span>plotSmear<span style="color: #00169a">(</span>d.PS<span style="color: #00169a">, </span>panel.first<span style="color: #00169a">=</span>grid<span style="color: #00169a">(),</span>smooth.scatter<span style="color: #00169a">=</span><span style="color: #c68800">FALSE</span><span style="color: #00169a">,</span>main<span style="color: #00169a">=</span><span style="color: #b31100">'after TMM'</span><span style="color: #00169a">)</span></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; ">ma.plot<span style="color: #00169a">(</span>b<span style="color: #00169a">$</span>A<span style="color: #00169a">,</span>b<span style="color: #00169a">$</span>M<span style="color: #00169a">,</span>plot.method<span style="color: #00169a">=</span><span style="color: #b31100">'add'</span><span style="color: #00169a">,</span>cex<span style="color: #00169a">=</span><span style="color: #005218">0</span><span style="color: #00169a">)</span></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; ">rm<span style="color: #00169a">(</span>b<span style="color: #00169a">)</span></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; ">d.PS.2<span style="color: #00169a"> <- </span>calcNormFactors<span style="color: #00169a">(</span>PScounts<span style="color: #00169a">,</span>method<span style="color: #00169a">=</span><span style="color: #b31100">'RLE'</span><span style="color: #00169a">)</span></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; ">b<span style="color: #00169a"><-</span>plotSmear<span style="color: #00169a">(</span>d.PS<span style="color: #00169a">, </span>panel.first<span style="color: #00169a">=</span>grid<span style="color: #00169a">(),</span>smooth.scatter<span style="color: #00169a">=</span><span style="color: #c68800">FALSE</span><span style="color: #00169a">,</span>main<span style="color: #00169a">=</span><span style="color: #b31100">'after RLE'</span><span style="color: #00169a">)</span></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; ">ma.plot<span style="color: #00169a">(</span>b<span style="color: #00169a">$</span>A<span style="color: #00169a">,</span>b<span style="color: #00169a">$</span>M<span style="color: #00169a">,</span>plot.method<span style="color: #00169a">=</span><span style="color: #b31100">'add'</span><span style="color: #00169a">,</span>cex<span style="color: #00169a">=</span><span style="color: #005218">0</span><span style="color: #00169a">)</span></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; ">rm<span style="color: #00169a">(</span>b<span style="color: #00169a">)</span></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; ">d.PS.3<span style="color: #00169a"> <- </span>calcNormFactors<span style="color: #00169a">(</span>PScounts<span style="color: #00169a">,</span>method<span style="color: #00169a">=</span><span style="color: #b31100">'quantile'</span><span style="color: #00169a">)</span></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; ">b<span style="color: #00169a"><-</span>plotSmear<span style="color: #00169a">(</span>d.PS.3<span style="color: #00169a">, </span>panel.first<span style="color: #00169a">=</span>grid<span style="color: #00169a">(),</span>smooth.scatter<span style="color: #00169a">=</span><span style="color: #c68800">FALSE</span><span style="color: #00169a">,</span>main<span style="color: #00169a">=</span><span style="color: #b31100">'after quantile'</span><span style="color: #00169a">)</span></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; ">ma.plot<span style="color: #00169a">(</span>b<span style="color: #00169a">$</span>A<span style="color: #00169a">,</span>b<span style="color: #00169a">$</span>M<span style="color: #00169a">,</span>plot.method<span style="color: #00169a">=</span><span style="color: #b31100">'add'</span><span style="color: #00169a">,</span>cex<span style="color: #00169a">=</span><span style="color: #005218">0</span><span style="color: #00169a">)</span></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; ">rm<span style="color: #00169a">(</span>b<span style="color: #00169a">)</span></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Monaco; ">dev.off<span style="color: #00169a">()</span></div><div><font class="Apple-style-span" color="#00169A"><br></font></div><div><font class="Apple-style-span" color="#00169A"><div>> d.PS$sample ###(after TMM)</div><div> files group Patient lib.size norm.factors</div><div>LS-25 LS252.counts.txt LS 25 23067191 0.9085</div><div>LS-28 LS287.counts.txt LS 28 20684675 0.9056</div><div>LS-29 LS292.counts.txt LS 29 19881245 0.9965</div><div>NL-25 NL251.counts.txt NL 25 19665929 1.0129</div><div>NL-28 NL286.counts.txt NL 28 22938039 1.1554</div><div>NL-29 NL291.counts.txt NL 29 20541691 1.0422</div><div>> </div><div>> d.PS.2$sample ###after RLE</div><div> files group Patient lib.size norm.factors</div><div>LS-25 LS252.counts.txt LS 25 23067191 0.9495</div><div>LS-28 LS287.counts.txt LS 28 20684675 0.9898</div><div>LS-29 LS292.counts.txt LS 29 19881245 1.0385</div><div>NL-25 NL251.counts.txt NL 25 19665929 0.9592</div><div>NL-28 NL286.counts.txt NL 28 22938039 1.0572</div><div>NL-29 NL291.counts.txt NL 29 20541691 1.0104</div><div><br></div><div>> d.PS.3$sample ###after quantiles</div><div> files group Patient lib.size norm.factors</div><div>LS-25 LS252.counts.txt LS 25 23067191 0.8659</div><div>LS-28 LS287.counts.txt LS 28 20684675 0.9656</div><div>LS-29 LS292.counts.txt LS 29 19881245 1.1302</div><div>NL-25 NL251.counts.txt NL 25 19665929 0.8887</div><div>NL-28 NL286.counts.txt NL 28 22938039 1.0885</div><div>NL-29 NL291.counts.txt NL 29 20541691 1.0939</div></font></div><div><font class="Apple-style-span" color="#00169A"><br></font></div><div><font class="Apple-style-span" color="#00169A"></font></div></div></font></font></div></div></div></div></span></div></span></div></div></body></html>