From 1c0b2dd3e1ef06ea05c200a4c3aa624cacca4f0c Mon Sep 17 00:00:00 2001 From: Michel RAYMOND <13813708+michelraymond@users.noreply.github.com> Date: Sun, 29 Oct 2017 19:11:10 +0100 Subject: [PATCH 1/5] new analysis --- analysis.1412/DEanalysis.R | 48 + analysis.1412/ERR990557s.counts | 17486 ++++++++++++++++++ analysis.1412/ERR990558s.counts | 17486 ++++++++++++++++++ analysis.1412/ERR990559s.counts | 17486 ++++++++++++++++++ analysis.1412/ERR990560s.counts | 17486 ++++++++++++++++++ analysis.1412/GeneList.csv | 1605 ++ analysis.1412/README.md | 67 + analysis.1412/graphs/DispersionEst-plot.png | Bin 0 -> 42250 bytes analysis.1412/graphs/MA-plot.png | Bin 0 -> 17027 bytes analysis.1412/graphs/PCA-plot.png | Bin 0 -> 10108 bytes 10 files changed, 71664 insertions(+) create mode 100755 analysis.1412/DEanalysis.R create mode 100755 analysis.1412/ERR990557s.counts create mode 100755 analysis.1412/ERR990558s.counts create mode 100755 analysis.1412/ERR990559s.counts create mode 100755 analysis.1412/ERR990560s.counts create mode 100644 analysis.1412/GeneList.csv create mode 100644 analysis.1412/README.md create mode 100644 analysis.1412/graphs/DispersionEst-plot.png create mode 100644 analysis.1412/graphs/MA-plot.png create mode 100644 analysis.1412/graphs/PCA-plot.png diff --git a/analysis.1412/DEanalysis.R b/analysis.1412/DEanalysis.R new file mode 100755 index 0000000..e9b75b0 --- /dev/null +++ b/analysis.1412/DEanalysis.R @@ -0,0 +1,48 @@ +# Loading libraries +library(DESeq2) +library(data.table) + + +# Creation of sample table of counts files for DESeq2 ------------------------ + +countfiles = character(4) +samplename = character(4) +for (i in 1:4) { + countfiles[i] = sprintf("ERR9905%ds.counts", i + 56) + samplename[i] = sprintf("ERR9905%d", i + 56) +} +condition = factor(c("a", "a", "b", "b")) + +sampTable = data.table(sampName = samplename, file = countfiles, condition = condition) + + +# Differential gene expression analysis ------------------------------------- + +DEdataset = DESeqDataSetFromHTSeqCount(sampleTable = sampTable, design = ~condition) +dds = DESeq(DEdataset) +res = results(dds) + + +# Plots and table of differential expressions ------------------------------- + +png(filename="./graphs/MA-plot.png") +plotMA(dds, ylim=c(-8, 8)) +dev.off() + +png(filename="./graphs/DispersionEst-plot.png") +plotDispEsts(dds) +dev.off() + +png(filename="./graphs/PCA-plot.png") +plotPCA(DESeqTransform(dds)) +dev.off() + +restable = as.data.frame(res) +setDT(restable, keep.rownames = "geneName") + + +# Extraction of significantly (padj<0.01) differentially expressed genes ---- + +res_padj = restable[padj < 0.01][order(padj)] + +fwrite(res_padj[,"geneName"], "GeneList.csv", col.names = F) diff --git a/analysis.1412/ERR990557s.counts b/analysis.1412/ERR990557s.counts new file mode 100755 index 0000000..826ebed --- /dev/null +++ b/analysis.1412/ERR990557s.counts @@ -0,0 +1,17486 @@ +FBgn0000003 19 +FBgn0000008 373 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+FBgn0015774 +FBgn0029821 +FBgn0003942 +FBgn0026702 +FBgn0035160 +FBgn0039594 +FBgn0267326 +FBgn0267991 +FBgn0266719 +FBgn0034883 +FBgn0283680 +FBgn0033133 +FBgn0263316 +FBgn0000277 +FBgn0010078 +FBgn0263396 +FBgn0032350 +FBgn0033092 +FBgn0003366 diff --git a/analysis.1412/README.md b/analysis.1412/README.md new file mode 100644 index 0000000..d84ce7e --- /dev/null +++ b/analysis.1412/README.md @@ -0,0 +1,67 @@ +# RNAseq report + +1. Download 4 sequence datasets deposited to the EBI [ENA](http://www.ebi.ac.uk/ena) +2. Extract fastq files +3. for each file, select 8,000,000 (8 millions) of sequence reads and generate the following sample files + +The 8M first reads were selected from the extracted and downloaded files using this bash one liner: + +```bash +for droso in {57..60}; do wget -qO- ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR990/ERR9905${droso}/ERR9905${droso}.fastq.gz | gunzip -c | head -n 32000000 > ERR9905${droso}s.fastq; done +``` + +It also allowed not to have to download the whole sequence files, only the first 8M reads needed for analysis. + +4. Align these read datasets to the reference genome by any appropriate mean, and generate a sorted bam alignment file. + +Reference genome for the Drosophila melanogaster was downloaded from Flybase (r6.17 at the time of the analysis): + +```bash +wget -qO- ftp://ftp.flybase.net/genomes/Drosophila_melanogaster/dmel_r6.17_FB2017_04/fasta/dmel-all-chromosome-r6.17.fasta.gz | gunzip -c > ./dmel/dmel.fa +``` + +Genome's index files were then generated using Bowtie 2 (v2.3.3.1) tool: + +```bash +./bowtie2-2.3.3.1-linux-x86_64/bowtie2-build ./dmel/dmel.fa ./dmel/dmel +``` + +The reads datasets were thus aligned to D.melanogaster's genome using TopHat (v2.1.1) tool, results were stored in separate folders named accordingly for each dataset: + +```bash +for droso in {57..60}; do ./tophat-2.1.1.Linux_x86_64/tophat -o ./ERR9905${droso} -p 4 ./dmel/dmel ERR9905${droso}s.fastq; done +``` + +The generated BAM files were then sorted (using the default sorting, by position) and indexed using SAMtools (v0.1.18, included in the TopHat package): + +```bash +for droso in {57..60}; do ./tophat-2.1.1.Linux_x86_64/samtools_0.1.18 sort ./ERR9905${droso}/accepted_hits.bam ./ERR9905${droso}/dmel_sorted; ./tophat-2.1.1.Linux_x86_64/samtools_0.1.18 index ./ERR9905${droso}/dmel_sorted.bam; done +``` + +Four separate dmel_sorted.bam files were thus generated from this section, placed in each reads dataset's respective folder. + +5. Count reads aligning to genome's genes by any appropriate mean + +Reads per genome's genes were counted using HTSeq (v0.8.0) tool, specifically the _htseq-count_ included script, and using the corresponding GTF file downloaded from Flybase (ftp://ftp.flybase.net/genomes/Drosophila_melanogaster/dmel_r6.17_FB2017_04/gtf/dmel-all-r6.17.gtf.gz), thus generating separate .counts files for each reads dataset containing the number of reads for each gene of D. melanogaster.: + +```bash +for droso in {57..60}; do htseq-count -f bam -r pos ./ERR9905${droso}/dmel_sorted.bam dmel-all-r6.17.gtf.gz > ERR9905${droso}s.counts; done +``` + +Generated counts files are presented in the current folder for use as input for the differential expression analysis. + +6. Perform a statistical differential expression analysis and report using any appropriate figure(s)/graph(s) +7. select a list of genes likely to be differentially expressed with a p-adj value < 0.01 +8. Code a simple script that parse the table of differential expressions (from *6.*) and return the genes with a p-adj value < 0.01 for rejection of H0 (non differential expression) + +Cf R script for details on the analysis. + +Graphs generated by the script include an MA plot: + +![Alt text](/graphs/MA-plot.png?raw=true "Optional Title") + +And a plot of the dispersion estimates: + +![Alt text](/graphs/DispersionEst-plot.png?raw=true "Optional Title2") + +A list of significantly (padj < 0.01) differentially expressed genes, generated by the same R script, can be found in the GeneList.csv file. \ No newline at end of file diff --git a/analysis.1412/graphs/DispersionEst-plot.png b/analysis.1412/graphs/DispersionEst-plot.png new file mode 100644 index 0000000000000000000000000000000000000000..ebd6e8c2109de0ef9484729aae33011e64bdbb7d GIT binary patch literal 42250 zcmeEu^;?utxAp)6(t}T(_?phP3rXq`jNsfs?AaLa6o@gKtNDKdd-$sV-9MmwkArLeO 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zWC}A(H~#jOe6Gm8mzSs#c?n8L+H=)PPVOt@-42e96xc#-Z70awAQvqM?+0XV1R)|^ zthDpgh@I3Mo@=6ba`GnUU48vch(@~!SYHksK1`m>%TUiPh0zspd)eI05%seEme%gT}hZZ}80)4d$ zmANtH8EnauCZ%BvE4T%#1Z;|d`$|twhoXn_Xqj~SVbxwm*oF3R&(CD!ymTiFfUjxOxX-9xS+uq!G!+(7|C5V%38De3zM@Lv~UY&mJ(aojB18 zcndcIGBOR1$IAu5H!$%?!94IX8eIXQ<|a;4Q#0~!=Pa=5l}H;cLqluPsO26|R-oP> z?gLN*sSIpW4>g~9Cu(?z#9S=8j|=kyJ{1Z?HA*xEd|zp)2h<3Jj-)<^V9M(d08wb@ z=vq+1%TwLc+Z#*ukmNxRfwqSMzOM^L?pPz?e6gbl6@js0c)Xf#o@i}l4w(+L(b<{WLcZiW-LIQ&?GQhxI46(aIg*d$@MP_fd z2&Ht*W#*o@JuDlyq|MAg#TJZtA>G>w#BL8QZn)*E!p^;x9QXi9J8&-SVIIg!K-;*> zCUSuKFz$h9ObV<;srMklDW Date: Sun, 29 Oct 2017 19:19:25 +0100 Subject: [PATCH 2/5] readme corrections --- analysis.1412/README.md | 20 ++++++++++---------- 1 file changed, 10 insertions(+), 10 deletions(-) diff --git a/analysis.1412/README.md b/analysis.1412/README.md index d84ce7e..351ee7a 100644 --- a/analysis.1412/README.md +++ b/analysis.1412/README.md @@ -1,8 +1,8 @@ # RNAseq report -1. Download 4 sequence datasets deposited to the EBI [ENA](http://www.ebi.ac.uk/ena) -2. Extract fastq files -3. for each file, select 8,000,000 (8 millions) of sequence reads and generate the following sample files +> 1. Download 4 sequence datasets deposited to the EBI [ENA](http://www.ebi.ac.uk/ena) +> 2. Extract fastq files +> 3. for each file, select 8,000,000 (8 millions) of sequence reads and generate the following sample files The 8M first reads were selected from the extracted and downloaded files using this bash one liner: @@ -12,7 +12,7 @@ for droso in {57..60}; do wget -qO- ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR990/ER It also allowed not to have to download the whole sequence files, only the first 8M reads needed for analysis. -4. Align these read datasets to the reference genome by any appropriate mean, and generate a sorted bam alignment file. +> 4. Align these read datasets to the reference genome by any appropriate mean, and generate a sorted bam alignment file. Reference genome for the Drosophila melanogaster was downloaded from Flybase (r6.17 at the time of the analysis): @@ -40,7 +40,7 @@ for droso in {57..60}; do ./tophat-2.1.1.Linux_x86_64/samtools_0.1.18 sort ./ERR Four separate dmel_sorted.bam files were thus generated from this section, placed in each reads dataset's respective folder. -5. Count reads aligning to genome's genes by any appropriate mean +> 5. Count reads aligning to genome's genes by any appropriate mean Reads per genome's genes were counted using HTSeq (v0.8.0) tool, specifically the _htseq-count_ included script, and using the corresponding GTF file downloaded from Flybase (ftp://ftp.flybase.net/genomes/Drosophila_melanogaster/dmel_r6.17_FB2017_04/gtf/dmel-all-r6.17.gtf.gz), thus generating separate .counts files for each reads dataset containing the number of reads for each gene of D. melanogaster.: @@ -50,18 +50,18 @@ for droso in {57..60}; do htseq-count -f bam -r pos ./ERR9905${droso}/dmel_sorte Generated counts files are presented in the current folder for use as input for the differential expression analysis. -6. Perform a statistical differential expression analysis and report using any appropriate figure(s)/graph(s) -7. select a list of genes likely to be differentially expressed with a p-adj value < 0.01 -8. Code a simple script that parse the table of differential expressions (from *6.*) and return the genes with a p-adj value < 0.01 for rejection of H0 (non differential expression) +> 6. Perform a statistical differential expression analysis and report using any appropriate figure(s)/graph(s) +> 7. select a list of genes likely to be differentially expressed with a p-adj value < 0.01 +> 8. Code a simple script that parse the table of differential expressions (from *6.*) and return the genes with a p-adj value < 0.01 for rejection of H0 (non differential expression) Cf R script for details on the analysis. Graphs generated by the script include an MA plot: -![Alt text](/graphs/MA-plot.png?raw=true "Optional Title") +![Alt text](/analysis.1412/graphs/MA-plot.png?raw=true "Optional Title") And a plot of the dispersion estimates: -![Alt text](/graphs/DispersionEst-plot.png?raw=true "Optional Title2") +![Alt text](/analysis.1412/graphs/DispersionEst-plot.png?raw=true "Optional Title2") A list of significantly (padj < 0.01) differentially expressed genes, generated by the same R script, can be found in the GeneList.csv file. \ No newline at end of file From 1284280535700f78ffe763ea74357abea313cafe Mon Sep 17 00:00:00 2001 From: Michel RAYMOND <13813708+michelraymond@users.noreply.github.com> Date: Sun, 29 Oct 2017 19:25:28 +0100 Subject: [PATCH 3/5] readme corrections 2 --- analysis.1412/README.md | 6 +++--- 1 file changed, 3 insertions(+), 3 deletions(-) diff --git a/analysis.1412/README.md b/analysis.1412/README.md index 351ee7a..466af85 100644 --- a/analysis.1412/README.md +++ b/analysis.1412/README.md @@ -4,7 +4,7 @@ > 2. Extract fastq files > 3. for each file, select 8,000,000 (8 millions) of sequence reads and generate the following sample files -The 8M first reads were selected from the extracted and downloaded files using this bash one liner: +The first 8 millions reads were selected from the extracted and downloaded files using this bash one liner: ```bash for droso in {57..60}; do wget -qO- ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR990/ERR9905${droso}/ERR9905${droso}.fastq.gz | gunzip -c | head -n 32000000 > ERR9905${droso}s.fastq; done @@ -58,10 +58,10 @@ Cf R script for details on the analysis. Graphs generated by the script include an MA plot: -![Alt text](/analysis.1412/graphs/MA-plot.png?raw=true "Optional Title") +![MA plot](/analysis.1412/graphs/MA-plot.png?raw=true "MA plot") And a plot of the dispersion estimates: -![Alt text](/analysis.1412/graphs/DispersionEst-plot.png?raw=true "Optional Title2") +![Dispersion estimate plot](/analysis.1412/graphs/DispersionEst-plot.png?raw=true "Dispersion estimate plot") A list of significantly (padj < 0.01) differentially expressed genes, generated by the same R script, can be found in the GeneList.csv file. \ No newline at end of file From 9acd5b6d0ca2a867d13ced871b0dcdc345300413 Mon Sep 17 00:00:00 2001 From: Michel RAYMOND <13813708+michelraymond@users.noreply.github.com> Date: Sun, 29 Oct 2017 20:11:26 +0100 Subject: [PATCH 4/5] added alignment logs and summary files --- analysis.1412/README.md | 4 +- .../alignment/align_summary_ERR990557.txt | 5 + .../alignment/align_summary_ERR990558.txt | 5 + .../alignment/align_summary_ERR990559.txt | 5 + .../alignment/align_summary_ERR990560.txt | 5 + analysis.1412/alignment/typescript.log | 297 ++++++++++++++++++ 6 files changed, 320 insertions(+), 1 deletion(-) create mode 100755 analysis.1412/alignment/align_summary_ERR990557.txt create mode 100755 analysis.1412/alignment/align_summary_ERR990558.txt create mode 100755 analysis.1412/alignment/align_summary_ERR990559.txt create mode 100755 analysis.1412/alignment/align_summary_ERR990560.txt create mode 100755 analysis.1412/alignment/typescript.log diff --git a/analysis.1412/README.md b/analysis.1412/README.md index 466af85..32fe013 100644 --- a/analysis.1412/README.md +++ b/analysis.1412/README.md @@ -32,6 +32,8 @@ The reads datasets were thus aligned to D.melanogaster's genome using TopHat (v2 for droso in {57..60}; do ./tophat-2.1.1.Linux_x86_64/tophat -o ./ERR9905${droso} -p 4 ./dmel/dmel ERR9905${droso}s.fastq; done ``` +Terminal logs for alignment and summary files are included in the ./alignment folder. + The generated BAM files were then sorted (using the default sorting, by position) and indexed using SAMtools (v0.1.18, included in the TopHat package): ```bash @@ -42,7 +44,7 @@ Four separate dmel_sorted.bam files were thus generated from this section, place > 5. Count reads aligning to genome's genes by any appropriate mean -Reads per genome's genes were counted using HTSeq (v0.8.0) tool, specifically the _htseq-count_ included script, and using the corresponding GTF file downloaded from Flybase (ftp://ftp.flybase.net/genomes/Drosophila_melanogaster/dmel_r6.17_FB2017_04/gtf/dmel-all-r6.17.gtf.gz), thus generating separate .counts files for each reads dataset containing the number of reads for each gene of D. melanogaster.: +Reads per genome's genes were counted using HTSeq (v0.8.0) tool, specifically the _htseq-count_ script included, and using the corresponding GTF file downloaded from Flybase (ftp://ftp.flybase.net/genomes/Drosophila_melanogaster/dmel_r6.17_FB2017_04/gtf/dmel-all-r6.17.gtf.gz), thus generating separate .counts files for each reads dataset containing the number of reads for each gene of D. melanogaster.: ```bash for droso in {57..60}; do htseq-count -f bam -r pos ./ERR9905${droso}/dmel_sorted.bam dmel-all-r6.17.gtf.gz > ERR9905${droso}s.counts; done diff --git a/analysis.1412/alignment/align_summary_ERR990557.txt b/analysis.1412/alignment/align_summary_ERR990557.txt new file mode 100755 index 0000000..98430ef --- /dev/null +++ b/analysis.1412/alignment/align_summary_ERR990557.txt @@ -0,0 +1,5 @@ +Reads: + Input : 8000000 + Mapped : 6003735 (75.0% of input) + of these: 849706 (14.2%) have multiple alignments (44 have >20) +75.0% overall read mapping rate. diff --git a/analysis.1412/alignment/align_summary_ERR990558.txt b/analysis.1412/alignment/align_summary_ERR990558.txt new file mode 100755 index 0000000..e88227a --- /dev/null +++ b/analysis.1412/alignment/align_summary_ERR990558.txt @@ -0,0 +1,5 @@ +Reads: + Input : 8000000 + Mapped : 6874261 (85.9% of input) + of these: 922346 (13.4%) have multiple alignments (69 have >20) +85.9% overall read mapping rate. diff --git a/analysis.1412/alignment/align_summary_ERR990559.txt b/analysis.1412/alignment/align_summary_ERR990559.txt new file mode 100755 index 0000000..09ec034 --- /dev/null +++ b/analysis.1412/alignment/align_summary_ERR990559.txt @@ -0,0 +1,5 @@ +Reads: + Input : 8000000 + Mapped : 6412816 (80.2% of input) + of these: 1493766 (23.3%) have multiple alignments (86 have >20) +80.2% overall read mapping rate. diff --git a/analysis.1412/alignment/align_summary_ERR990560.txt b/analysis.1412/alignment/align_summary_ERR990560.txt new file mode 100755 index 0000000..8ee3e67 --- /dev/null +++ b/analysis.1412/alignment/align_summary_ERR990560.txt @@ -0,0 +1,5 @@ +Reads: + Input : 8000000 + Mapped : 7030248 (87.9% of input) + of these: 1277125 (18.2%) have multiple alignments (74 have >20) +87.9% overall read mapping rate. diff --git a/analysis.1412/alignment/typescript.log b/analysis.1412/alignment/typescript.log new file mode 100755 index 0000000..2618a31 --- /dev/null +++ b/analysis.1412/alignment/typescript.log @@ -0,0 +1,297 @@ +Script started on 2017-10-29 17:56:08 + +someone@somewhere:~/rnaseq$ ./bowtie2-2.3.3.1-linux-x86_64/bowtie2-build ./dmel/dmel.fa ./dmel/dmel +Settings: + Output files: "./dmel/dmel.*.bt2" + Line rate: 6 (line is 64 bytes) + Lines per side: 1 (side is 64 bytes) + Offset rate: 4 (one in 16) + FTable chars: 10 + Strings: unpacked + Max bucket size: default + Max bucket size, sqrt multiplier: default + Max bucket size, len divisor: 4 + Difference-cover sample period: 1024 + Endianness: little + Actual local endianness: little + Sanity checking: disabled + Assertions: disabled + Random seed: 0 + Sizeofs: void*:8, int:4, long:8, size_t:8 +Input files DNA, FASTA: + ./dmel/dmel.fa +Building a SMALL index +Reading reference sizes + Time reading reference sizes: 00:00:02 +Calculating joined length +Writing header +Reserving space for joined string +Joining reference sequences + Time to join reference sequences: 00:00:01 +bmax according to bmaxDivN setting: 35643256 +Using parameters --bmax 26732442 --dcv 1024 + Doing ahead-of-time memory usage test + Passed! Constructing with these parameters: --bmax 26732442 --dcv 1024 +Constructing suffix-array element generator +Building DifferenceCoverSample + Building sPrime + Building sPrimeOrder + V-Sorting samples + V-Sorting samples time: 00:00:04 + Allocating rank array + Ranking v-sort output + Ranking v-sort output time: 00:00:00 + Invoking Larsson-Sadakane on ranks + Invoking Larsson-Sadakane on ranks time: 00:00:02 + Sanity-checking and returning +Building samples +Reserving space for 12 sample suffixes +Generating random suffixes +QSorting 12 sample offsets, eliminating duplicates +QSorting sample offsets, eliminating duplicates time: 00:00:00 +Multikey QSorting 12 samples + (Using difference cover) + Multikey QSorting samples time: 00:00:00 +Calculating bucket sizes +Splitting and merging + Splitting and merging time: 00:00:00 +Avg bucket size: 1.42573e+08 (target: 26732441) +Converting suffix-array elements to index image +Allocating ftab, absorbFtab +Entering Ebwt loop +Getting block 1 of 1 + No samples; assembling all-inclusive block + Sorting block of length 142573024 for bucket 1 + (Using difference cover) + Sorting block time: 00:01:07 +Returning block of 142573025 for bucket 1 +Exited Ebwt loop +fchr[A]: 0 +fchr[C]: 41351058 +fchr[G]: 71311730 +fchr[T]: 101240543 +fchr[$]: 142573024 +Exiting Ebwt::buildToDisk() +Returning from initFromVector +Wrote 52172432 bytes to primary EBWT file: ./dmel/dmel.1.bt2 +Wrote 35643264 bytes to secondary EBWT file: ./dmel/dmel.2.bt2 +Re-opening _in1 and _in2 as input streams +Returning from Ebwt constructor +Headers: + len: 142573024 + bwtLen: 142573025 + sz: 35643256 + bwtSz: 35643257 + lineRate: 6 + offRate: 4 + offMask: 0xfffffff0 + ftabChars: 10 + eftabLen: 20 + eftabSz: 80 + ftabLen: 1048577 + ftabSz: 4194308 + offsLen: 8910815 + offsSz: 35643260 + lineSz: 64 + sideSz: 64 + sideBwtSz: 48 + sideBwtLen: 192 + numSides: 742568 + numLines: 742568 + ebwtTotLen: 47524352 + ebwtTotSz: 47524352 + color: 0 + reverse: 0 +Total time for call to driver() for forward index: 00:01:26 +Reading reference sizes + Time reading reference sizes: 00:00:01 +Calculating joined length +Writing header +Reserving space for joined string +Joining reference sequences + Time to join reference sequences: 00:00:01 + Time to reverse reference sequence: 00:00:00 +bmax according to bmaxDivN setting: 35643256 +Using parameters --bmax 26732442 --dcv 1024 + Doing ahead-of-time memory usage test + Passed! Constructing with these parameters: --bmax 26732442 --dcv 1024 +Constructing suffix-array element generator +Building DifferenceCoverSample + Building sPrime + Building sPrimeOrder + V-Sorting samples + V-Sorting samples time: 00:00:04 + Allocating rank array + Ranking v-sort output + Ranking v-sort output time: 00:00:01 + Invoking Larsson-Sadakane on ranks + Invoking Larsson-Sadakane on ranks time: 00:00:01 + Sanity-checking and returning +Building samples +Reserving space for 12 sample suffixes +Generating random suffixes +QSorting 12 sample offsets, eliminating duplicates +QSorting sample offsets, eliminating duplicates time: 00:00:00 +Multikey QSorting 12 samples + (Using difference cover) + Multikey QSorting samples time: 00:00:00 +Calculating bucket sizes +Splitting and merging + Splitting and merging time: 00:00:00 +Avg bucket size: 1.42573e+08 (target: 26732441) +Converting suffix-array elements to index image +Allocating ftab, absorbFtab +Entering Ebwt loop +Getting block 1 of 1 + No samples; assembling all-inclusive block + Sorting block of length 142573024 for bucket 1 + (Using difference cover) + Sorting block time: 00:01:07 +Returning block of 142573025 for bucket 1 +Exited Ebwt loop +fchr[A]: 0 +fchr[C]: 41351058 +fchr[G]: 71311730 +fchr[T]: 101240543 +fchr[$]: 142573024 +Exiting Ebwt::buildToDisk() +Returning from initFromVector +Wrote 52172432 bytes to primary EBWT file: ./dmel/dmel.rev.1.bt2 +Wrote 35643264 bytes to secondary EBWT file: ./dmel/dmel.rev.2.bt2 +Re-opening _in1 and _in2 as input streams +Returning from Ebwt constructor +Headers: + len: 142573024 + bwtLen: 142573025 + sz: 35643256 + bwtSz: 35643257 + lineRate: 6 + offRate: 4 + offMask: 0xfffffff0 + ftabChars: 10 + eftabLen: 20 + eftabSz: 80 + ftabLen: 1048577 + ftabSz: 4194308 + offsLen: 8910815 + offsSz: 35643260 + lineSz: 64 + sideSz: 64 + sideBwtSz: 48 + sideBwtLen: 192 + numSides: 742568 + numLines: 742568 + ebwtTotLen: 47524352 + ebwtTotSz: 47524352 + color: 0 + reverse: 1 +Total time for backward call to driver() for mirror index: 00:01:25 + +someone@somewhere:~/rnaseq$ for droso in {57..60}; do ./tophat-2.1.1.Linux_x86_64/tophat -o ./ERR9905${droso} -p 4 ./dmel/dmel ERR9905${droso}s.fastq; done + + +[2017-10-29 18:00:43] Beginning TopHat run (v2.1.1) +----------------------------------------------- +[2017-10-29 18:00:43] Checking for Bowtie + Bowtie version: 2.3.3.1 +[2017-10-29 18:00:43] Checking for Bowtie index files (genome).. +[2017-10-29 18:00:43] Checking for reference FASTA file +[2017-10-29 18:00:43] Generating SAM header for ./dmel/dmel +[2017-10-29 18:00:43] Preparing reads + left reads: min. length=51, max. length=51, 7994925 kept reads (5075 discarded) +[2017-10-29 18:02:09] Mapping left_kept_reads to genome dmel with Bowtie2 +[2017-10-29 18:10:10] Mapping left_kept_reads_seg1 to genome dmel with Bowtie2 (1/2) +[2017-10-29 18:11:14] Mapping left_kept_reads_seg2 to genome dmel with Bowtie2 (2/2) +[2017-10-29 18:12:52] Searching for junctions via segment mapping + Coverage-search algorithm is turned on, making this step very slow + Please try running TopHat again with the option (--no-coverage-search) if this step takes too much time or memory. +[2017-10-29 18:19:18] Retrieving sequences for splices +[2017-10-29 18:19:41] Indexing splices +Building a SMALL index +[2017-10-29 18:20:21] Mapping left_kept_reads_seg1 to genome segment_juncs with Bowtie2 (1/2) +[2017-10-29 18:21:10] Mapping left_kept_reads_seg2 to genome segment_juncs with Bowtie2 (2/2) +[2017-10-29 18:22:23] Joining segment hits +[2017-10-29 18:22:53] Reporting output tracks +----------------------------------------------- +[2017-10-29 18:28:59] A summary of the alignment counts can be found in ./ERR990557/align_summary.txt +[2017-10-29 18:28:59] Run complete: 00:28:16 elapsed + +[2017-10-29 18:28:59] Beginning TopHat run (v2.1.1) +----------------------------------------------- +[2017-10-29 18:28:59] Checking for Bowtie + Bowtie version: 2.3.3.1 +[2017-10-29 18:28:59] Checking for Bowtie index files (genome).. +[2017-10-29 18:28:59] Checking for reference FASTA file +[2017-10-29 18:28:59] Generating SAM header for ./dmel/dmel +[2017-10-29 18:29:01] Preparing reads + left reads: min. length=51, max. length=51, 7992819 kept reads (7181 discarded) +[2017-10-29 18:30:31] Mapping left_kept_reads to genome dmel with Bowtie2 +[2017-10-29 18:38:03] Mapping left_kept_reads_seg1 to genome dmel with Bowtie2 (1/2) +[2017-10-29 18:38:45] Mapping left_kept_reads_seg2 to genome dmel with Bowtie2 (2/2) +[2017-10-29 18:39:56] Searching for junctions via segment mapping + Coverage-search algorithm is turned on, making this step very slow + Please try running TopHat again with the option (--no-coverage-search) if this step takes too much time or memory. +[2017-10-29 18:45:01] Retrieving sequences for splices +[2017-10-29 18:45:23] Indexing splices +Building a SMALL index +[2017-10-29 18:45:59] Mapping left_kept_reads_seg1 to genome segment_juncs with Bowtie2 (1/2) +[2017-10-29 18:46:35] Mapping left_kept_reads_seg2 to genome segment_juncs with Bowtie2 (2/2) +[2017-10-29 18:47:29] Joining segment hits +[2017-10-29 18:48:01] Reporting output tracks +----------------------------------------------- +[2017-10-29 18:54:53] A summary of the alignment counts can be found in ./ERR990558/align_summary.txt +[2017-10-29 18:54:53] Run complete: 00:25:53 elapsed + +[2017-10-29 18:54:53] Beginning TopHat run (v2.1.1) +----------------------------------------------- +[2017-10-29 18:54:53] Checking for Bowtie + Bowtie version: 2.3.3.1 +[2017-10-29 18:54:53] Checking for Bowtie index files (genome).. +[2017-10-29 18:54:53] Checking for reference FASTA file +[2017-10-29 18:54:53] Generating SAM header for ./dmel/dmel +[2017-10-29 18:54:55] Preparing reads + left reads: min. length=51, max. length=51, 7991531 kept reads (8469 discarded) +[2017-10-29 18:56:21] Mapping left_kept_reads to genome dmel with Bowtie2 +[2017-10-29 19:05:55] Mapping left_kept_reads_seg1 to genome dmel with Bowtie2 (1/2) +[2017-10-29 19:06:51] Mapping left_kept_reads_seg2 to genome dmel with Bowtie2 (2/2) +[2017-10-29 19:08:30] Searching for junctions via segment mapping + Coverage-search algorithm is turned on, making this step very slow + Please try running TopHat again with the option (--no-coverage-search) if this step takes too much time or memory. +[2017-10-29 19:13:59] Retrieving sequences for splices +[2017-10-29 19:14:21] Indexing splices +Building a SMALL index +[2017-10-29 19:15:00] Mapping left_kept_reads_seg1 to genome segment_juncs with Bowtie2 (1/2) +[2017-10-29 19:15:47] Mapping left_kept_reads_seg2 to genome segment_juncs with Bowtie2 (2/2) +[2017-10-29 19:16:55] Joining segment hits +[2017-10-29 19:17:33] Reporting output tracks +----------------------------------------------- +[2017-10-29 19:26:40] A summary of the alignment counts can be found in ./ERR990559/align_summary.txt +[2017-10-29 19:26:40] Run complete: 00:31:46 elapsed + +[2017-10-29 19:26:40] Beginning TopHat run (v2.1.1) +----------------------------------------------- +[2017-10-29 19:26:40] Checking for Bowtie + Bowtie version: 2.3.3.1 +[2017-10-29 19:26:40] Checking for Bowtie index files (genome).. +[2017-10-29 19:26:40] Checking for reference FASTA file +[2017-10-29 19:26:40] Generating SAM header for ./dmel/dmel +[2017-10-29 19:26:42] Preparing reads + left reads: min. length=51, max. length=51, 7994096 kept reads (5904 discarded) +[2017-10-29 19:28:08] Mapping left_kept_reads to genome dmel with Bowtie2 +[2017-10-29 19:36:08] Mapping left_kept_reads_seg1 to genome dmel with Bowtie2 (1/2) +[2017-10-29 19:36:46] Mapping left_kept_reads_seg2 to genome dmel with Bowtie2 (2/2) +[2017-10-29 19:38:01] Searching for junctions via segment mapping + Coverage-search algorithm is turned on, making this step very slow + Please try running TopHat again with the option (--no-coverage-search) if this step takes too much time or memory. +[2017-10-29 19:43:02] Retrieving sequences for splices +[2017-10-29 19:43:22] Indexing splices +Building a SMALL index +[2017-10-29 19:43:57] Mapping left_kept_reads_seg1 to genome segment_juncs with Bowtie2 (1/2) +[2017-10-29 19:44:30] Mapping left_kept_reads_seg2 to genome segment_juncs with Bowtie2 (2/2) +[2017-10-29 19:45:13] Joining segment hits +[2017-10-29 19:45:43] Reporting output tracks +----------------------------------------------- +[2017-10-29 19:53:35] A summary of the alignment counts can be found in ./ERR990560/align_summary.txt +[2017-10-29 19:53:35] Run complete: 00:26:54 elapsed + +Script done on 2017-10-29 19:55:30+0100 From 96d65999272495017f7c89a8f07c4442057954c0 Mon Sep 17 00:00:00 2001 From: Michel RAYMOND <13813708+michelraymond@users.noreply.github.com> Date: Tue, 31 Oct 2017 11:55:42 +0100 Subject: [PATCH 5/5] changed readme (fastqc+typos) --- analysis.1412/README.md | 8 ++++++-- 1 file changed, 6 insertions(+), 2 deletions(-) diff --git a/analysis.1412/README.md b/analysis.1412/README.md index 32fe013..2201915 100644 --- a/analysis.1412/README.md +++ b/analysis.1412/README.md @@ -12,6 +12,8 @@ for droso in {57..60}; do wget -qO- ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR990/ER It also allowed not to have to download the whole sequence files, only the first 8M reads needed for analysis. +FastQC software was then used to check for reads quality. Overall quality was good and no leftover adapter sequences from the Illumina sequencer was found. + > 4. Align these read datasets to the reference genome by any appropriate mean, and generate a sorted bam alignment file. Reference genome for the Drosophila melanogaster was downloaded from Flybase (r6.17 at the time of the analysis): @@ -56,7 +58,7 @@ Generated counts files are presented in the current folder for use as input for > 7. select a list of genes likely to be differentially expressed with a p-adj value < 0.01 > 8. Code a simple script that parse the table of differential expressions (from *6.*) and return the genes with a p-adj value < 0.01 for rejection of H0 (non differential expression) -Cf R script for details on the analysis. +Cf DEanalysis.R script for details on the analysis. Graphs generated by the script include an MA plot: @@ -66,4 +68,6 @@ And a plot of the dispersion estimates: ![Dispersion estimate plot](/analysis.1412/graphs/DispersionEst-plot.png?raw=true "Dispersion estimate plot") -A list of significantly (padj < 0.01) differentially expressed genes, generated by the same R script, can be found in the GeneList.csv file. \ No newline at end of file +Other graphs can be found in the ./graphs subfolder. + +A list of significantly (padj < 0.01) differentially expressed genes, generated by the abovementioned R script, can be found in the GeneList.csv file. \ No newline at end of file