small fixes

Merge branch 'main' of https://github.com/ncgr/DE-Bookdown

# Conflicts:
#	packages.bib

Author: evanizer8

.DS_Store

@@ -7,7 +7,7 @@
 2. Type the following on your command line, substituting in your username for <username>.
 
 ```
-ssh -p2310 <username>@inbre.ncgr.org
+ssh -p2311 <username>@inbre.ncgr.org
 ```
 
 3. Enter your password.
@@ -35,25 +35,33 @@ In a Finder window, you would see this:
 
 ### Find the shell in system you’ll use to log into the NCGR’s server
 
-+ For **Windows**: search for MobaXterm from the start menu.
-    + It may be useful to drag the terminal icon to the **desktop** for easier access in the future.
++ For **Windows**: search for MobaXterm from the start menu
+
++ For **Mac**: search for "terminal" using spotlight (the magnifying glass in the top right of your mac screen)
 
-+ For **Mac**: search for "terminal" in the bar located in the Launchpad (rocket icon in the taskbar).
-    + It may be useful to drag the terminal icon into the **Dock** for easier access in the future.
 
 ### Log on to NCGR's server 
 
-Enter the following command to log on to logrus:
++ For **Windows**:
+Click "Sessioins," then "SSH" button
+    + Remote host: username@inbre.ncgr.org
+    + Specify Username: username
+    + Port: 2311
+    + click ok
+
++ For **Mac**:
+
+Enter the following command in the linux terminal to log on to the NCGR Server:
 
 + substitute **your** personal username in for "username"
 
 ```
-ssh -p2310 <username>@inbre.ncgr.org
+ssh -p2311 <username>@inbre.ncgr.org
 ```
 
 Notes:
 
-+ Because we’re logging in remotely, the -p option is required to specify port 2310. 
++ Because we’re logging in remotely, the -p option is required to specify port 2311. 
 + If you’re prompted to confirm the connection, say "yes", then enter your password.
 
 ### Now that I logged on, where am I?
@@ -62,7 +70,7 @@ You’re at the command line interface of NCGR's analysis server!
 
 To the left of the command prompt, you should see something like this: 
 
-+ agomez@metagenomics-2310:~$
++ agomez@de-2311:~$
 
 Command **output** is shown after the "##" in this document.
 
@@ -309,7 +317,7 @@ touch scp_test.txt
 + the last period means that the destination is your working directory
 
 ```
-scp -P 2310 <username>@inbre.ncgr.org:~/linuxc/scp_test.txt .
+scp -P 2311 <username>@inbre.ncgr.org:~/linuxc/scp_test.txt .
 ```
 
 Note: When you designate a port with secure copy (scp), you use a capital P.
@@ -322,7 +330,7 @@ You will be prompted for your NCGR Server password if not using MobaXterm.
 + Again, run the scp command from your **local** terminal window:
 
 ```
-scp -P 2310 scp_test.txt <username>@inbre.ncgr.org:~/linuxc
+scp -P 2311 scp_test.txt <username>@inbre.ncgr.org:~/linuxc
 ```
 
 6) Check to see if the file you copied from your computer is on **the NCGR Server**!
@@ -376,7 +384,7 @@ If you can't execute commands, then CTRL c
 ## ^C
 ```
 
-You should be returned to your prompt: username@metagenomics-2310:~/linuxc$
+You should be returned to your prompt: username@de-2311:~/linuxc$
 
 
 ## Linux basics: Part II
@@ -386,7 +394,7 @@ You should be returned to your prompt: username@metagenomics-2310:~/linuxc$
 #### Syntax: ln -s FileYouWantToLink/PointTo NameYouWantToGiveIt{-}
 
 ```
-ln -s /home/data/metagenomics-2310/covid.fasta covid.fasta
+ln -s /home/data/de-2311/covid.fasta covid.fasta
 
 ls -l
 ```
@@ -484,7 +492,7 @@ cat covid.fasta | tail
 Fastq files contain sequence reads and associated **meta data**.
 
 ```
-ln -s /home/data/metagenomics-2310/SP1.fq 
+ln -s /home/data/de-2311/SP1.fq 
 
 ls -ltr
 ```
@@ -686,7 +694,7 @@ Powerful Z commands (zcat)
 Let’s copy another file:
 
 ```
-cp /home/data/metagenomics-2310/table1.txt.gz .
+cp /home/data/de-2311/table1.txt.gz .
 zcat table1.txt.gz
 
 ```
@@ -978,7 +986,7 @@ There is also cheat sheet on the weebly site under Supplemental Documents.
 2) Enter the following command to log on to the NCGR Server: 
 
 ```
-ssh -p2310 <username>@inbre.ncgr.org
+ssh -p2311 <username>@inbre.ncgr.org
 ```
 
 + Don't forget to substitute **your** personal username in

02.Rmd

@@ -24,14 +24,14 @@ OPTION 1:
 ```{bash, eval=FALSE}
 source activate sra-tools
 
-while read line; do fasterq-dump --outdir ./ ${line}; done < /home/elavelle/SRRlist.txt
+while read line; do fasterq-dump --outdir ./ ${line}; done < /home/elavelle/DGE_workshop/SRRlist.txt
 
 ```
 
 
 OPTION 2:
 ```{bash, eval=FALSE}
-ln -s home/elavelle/DGE_workshop/reads/*.gz ./
+ln -s /home/elavelle/DGE_workshop/reads/*.gz ./
 ```
 
 ## fastp QC
@@ -41,12 +41,16 @@ conda deactivate
 
 source activate fastp
 
-for file in SRR*; do fastp -w 10 -i ${file} -o ${file}.fastp.gz; done
+for file in SRR*; do fastp -w 10 -h ${file}.html -i ${file} -o ${file}.fastp.gz; done
 ```
 This sort of messes up the extensions. Rename the files
 
 ```{bash, eval=FALSE}
 rename .gz.fastp.gz .fastp.gz *.gz
+
+OR
+
+while read line; do mv ${line}.fastq.gz.fastp.gz ${line}.fastq.fastp.gz; done < /home/elavelle/SRRlist.tx
 ```
 
 Download and view the .html report.
@@ -60,7 +64,7 @@ following example illustrates one use of this built-in variable.
 
 Prints the line number of the first 10 lines
 ```{bash, eval=FALSE}
-zcat SRR23869771.fastq.fastq.gz | awk '{print NR}' | head
+zcat SRR23869771.fastq.fastp.gz | awk '{print NR}' | head
 
 ```
 awk modulo arithmatic: The modulo operator, "%", returns the remainder of division. The expression

03QC.Rmd

@@ -26,14 +26,18 @@ wget https://ftp.ncbi.nlm.nih.gov/genomes/refseq/vertebrate_mammalian/Mus_muscul
 Activate environment, and use HISAT2 to create indices. If time prohibits, we will likely need to terminate and softlink to output files.
 
 ```{bash, eval=FALSE}
+gunzip GCF_000001635.27_GRCm39_genomic.fna.gz
+
 source activate hisat2
 
 hisat2-build -p 10 GCF_000001635.27_GRCm39_genomic.fna GRCm39.index
 ```
 
-From the reads directory, run alignments
+Make the output directory, then, trom the reads directory, run alignments
 
 ```{bash, eval=FALSE}
+mkdir ~/DGE_workshop/alignments
+
 for file in *.fastp.gz; do hisat2 -x /home/$USER/DGE_workshop/genome/GRCm39.index --threads 10 -U ${file} -S /home/$USER/DGE_workshop/alignments/${file}.sam; done
 ```
 
@@ -53,6 +57,9 @@ Some alignment tools (HISAT2 for example) will print alignment metrics after gen
 Change environments and use this statistics summarization command. It takes about a minute per sample.
 
 ```{bash, eval=FALSE}
+conda deactivate
+source activate samtools
+
 samtools flagstat SRR23869771.fastq.fastp.gz.sam
 ```
 
@@ -96,6 +103,8 @@ Therefore, the number of primary alignments can also be found by:
 samtools view -F 0x904 -c SRR23869771.fastq.fastp.gz.sam 
 ```
 
+Note that this is less than the total number of reads in our file. This operates in an "or" fashion. If we run the above command with "-f", it yields 0, because the postive search runs in an "and" fashion.
+
 Exercise: Select another sample, and using any of the means above, determine 1) Total read count 2) Total mapped reads 3) Total primary alignments
 
 ## .sam/.bam conversion and alignment sorting
@@ -107,7 +116,11 @@ for file in *.sam; do samtools view -u ${file} | samtools sort -o ${file}.sorted
 Rename the output files
 
 ```{bash, eval=FALSE}
-rename .fastq.fastp.gz.sam.sorted .sorted.bam *.sorted
+rename .fastq.fastp.gz.sam.sorted .sorted.bam *.bam 
+
+or
+
+file-rename -- 's/\.fastq.fastp.gz.sam.sorted$/.sorted.bam/' *.sorted
 ```
 
 Exercise: What average genome coverage do our primary alignments give us?

05Alignment.Rmd

@@ -15,10 +15,13 @@ Exercise: Which genome/annotation pair in the folder /home/elavelle/DGE_Virtual/
 
 # Generate counts form the alignments directory.
 
+Make sure the annotation is unzipped first.
+
 ```{bash, eval=FALSE}
+
 source activate featurecounts
 
-featureCounts -a ~/DGE_workshop/genome/GCF_000001635.27_GRCm39_genomic.gtf -o /home/$USER/DGE_workshop/count_matrix.tsv --largestOverlap -M --primary -T 10 -g gene *.bam
+featureCounts -a ~/DGE_workshop/genome/GCF_000001635.27_GRCm39_genomic.gff -o /home/$USER/DGE_workshop/count_matrix.tsv --largestOverlap -M --primary -T 10 -g gene *.bam
 ```
 
 FeatureCounts put in an extra row and some columns we want to get rid of before doing differential expression analysis.

06Abundancy_Estimation.Rmd

@@ -148,7 +148,7 @@ Sort our results table, and export a file with counts appended to it.
 ```{R, eval=FALSE}
 stress_from_control_sorted <- stress_from_control[order(stress_from_control$padj),]
 
-write.table(merge(as.data.frame(stress_from_control_sorted),as.data.frame(counts(dds,normalized=TRUE)), by="row.names"), row.names=TRUE, quote=FALSE, sep="\t", file="SfromC.tsv")
+write.table(merge(as.data.frame(stress_from_control_sorted),as.data.frame(counts(dds,normalized=TRUE)), by="row.names"), row.names=FALSE, quote=FALSE, sep="\t", file="SfromC.tsv")
 ```
 
 ## Heatmap
@@ -185,12 +185,27 @@ dev.off()
 Camk2n1 is skewing the color palette. Let's make a version omitting it.
 ```{R, eval=FALSE}
 pdf ("heatmapSigsMost.pdf")
-heatmap.2(plotCounts[-7,],  col=hmcol, cexRow=0.8,
+heatmap.2(plotCounts[-5,],  col=hmcol, cexRow=0.8,
           key.title = NA, scale="none", dendrogram="col", 
           trace="none", density.info='none', cexCol=0.6, margin=c(6,10))
 dev.off()
 ```
 
+If we want the same decreasing order:
+```{R, eval=FALSE}
+# Sorts, removes Camk2n1
+sorted <- row.names(data.frame(sort(rowMeans(plotCounts), decreasing=TRUE)))[-1]
+# makes new dataframe with these indeces
+new_counts <- plotCounts[match(sorted, row.names(plotCounts)),]
+
+pdf ("heatmapSigsMost.pdf")
+heatmap.2(new_counts,  col=hmcol, cexRow=0.8,
+          key.title = NA, scale="none", dendrogram="col", 
+          trace="none", density.info='none', cexCol=0.6, margin=c(6,10), Rowv=FALSE) 
+dev.off()
+```
+
+
 ![workflow](./Figures/heatmapSigsMost-1.png){width=100%}
 
 Principal Component Analysis now.
@@ -211,6 +226,8 @@ aesthetic layers
 x-axis labels and jittering
 
 ```{R, eval=FALSE}
+libary('ggplot2')
+
 for(k in 1:length(sigIndices)) {  
   gene <- rownames(stress_from_control_sorted)[k]
   pval <- format(stress_from_control_sorted$padj[k], digits=4)

07DGE.Rmd

@@ -157,6 +157,7 @@ _____
 Feedback Survey\
 
 Logrus Access Request\
+
 _____
 
 <span style="color: forestgreen;">Green</span> text indicates hands-on sessions. Please note that the timing might shift slightly.

index.Rmd

@@ -3,15 +3,23 @@ @Manual{R-base
   author = {{R Core Team}},
   organization = {R Foundation for Statistical Computing},
   address = {Vienna, Austria},
+<<<<<<< HEAD
   year = {2020},
+=======
+  year = {2022},
+>>>>>>> 2ac0111043520eeb6c18989debcc45a63bd62da7
   url = {https://www.R-project.org/},
 }
 
 @Manual{R-bookdown,
   title = {bookdown: Authoring Books and Technical Documents with R Markdown},
   author = {Yihui Xie},
   year = {2023},
+<<<<<<< HEAD
   note = {R package version 0.33},
+=======
+  note = {R package version 0.32},
+>>>>>>> 2ac0111043520eeb6c18989debcc45a63bd62da7
   url = {https://CRAN.R-project.org/package=bookdown},
 }
 
@@ -25,9 +33,13 @@ @Manual{R-knitr
 
 @Manual{R-rmarkdown,
   title = {rmarkdown: Dynamic Documents for R},
-  author = {JJ Allaire and Yihui Xie and Christophe Dervieux and Jonathan McPherson and Javier Luraschi and Kevin Ushey and Aron Atkins and Hadley Wickham and Joe Cheng and Winston Chang and Richard Iannone},
+  author = {JJ Allaire and Yihui Xie and Jonathan McPherson and Javier Luraschi and Kevin Ushey and Aron Atkins and Hadley Wickham and Joe Cheng and Winston Chang and Richard Iannone},
   year = {2023},
+<<<<<<< HEAD
   note = {R package version 2.21},
+=======
+  note = {R package version 2.20},
+>>>>>>> 2ac0111043520eeb6c18989debcc45a63bd62da7
   url = {https://CRAN.R-project.org/package=rmarkdown},
 }
 
@@ -68,7 +80,7 @@ @Book{rmarkdown2018
   publisher = {Chapman and Hall/CRC},
   address = {Boca Raton, Florida},
   year = {2018},
-  isbn = {9781138359338},
+  note = {ISBN 9781138359338},
   url = {https://bookdown.org/yihui/rmarkdown},
 }
 
@@ -78,7 +90,7 @@ @Book{rmarkdown2020
   publisher = {Chapman and Hall/CRC},
   address = {Boca Raton, Florida},
   year = {2020},
-  isbn = {9780367563837},
+  note = {ISBN 9780367563837},
   url = {https://bookdown.org/yihui/rmarkdown-cookbook},
 }