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 +FBgn0000014 218 +FBgn0000015 29 +FBgn0000017 432 +FBgn0000018 121 +FBgn0000022 0 +FBgn0000024 26 +FBgn0000028 0 +FBgn0000032 343 +FBgn0000036 3 +FBgn0000037 10 +FBgn0000038 0 +FBgn0000039 0 +FBgn0000042 6956 +FBgn0000043 1817 +FBgn0000044 3278 +FBgn0000045 36 +FBgn0000046 1995 +FBgn0000047 23 +FBgn0000052 799 +FBgn0000053 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0000000..2201915 --- /dev/null +++ b/analysis.1412/README.md @@ -0,0 +1,73 @@ +# 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 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${droso}s.fastq; done +``` + +It also allowed not to have to download the whole sequence files, only the first 8M reads needed for analysis. + +FastQC software was then used to check for reads quality. Overall quality was good and no leftover adapter sequences from the Illumina sequencer was found. + +> 4. Align these read datasets to the reference genome by any appropriate mean, and generate a sorted bam alignment file. + +Reference genome for the Drosophila melanogaster was downloaded from Flybase (r6.17 at the time of the analysis): + +```bash +wget -qO- ftp://ftp.flybase.net/genomes/Drosophila_melanogaster/dmel_r6.17_FB2017_04/fasta/dmel-all-chromosome-r6.17.fasta.gz | gunzip -c > ./dmel/dmel.fa +``` + +Genome's index files were then generated using Bowtie 2 (v2.3.3.1) tool: + +```bash +./bowtie2-2.3.3.1-linux-x86_64/bowtie2-build ./dmel/dmel.fa ./dmel/dmel +``` + +The reads datasets were thus aligned to D.melanogaster's genome using TopHat (v2.1.1) tool, results were stored in separate folders named accordingly for each dataset: + +```bash +for droso in {57..60}; do ./tophat-2.1.1.Linux_x86_64/tophat -o ./ERR9905${droso} -p 4 ./dmel/dmel ERR9905${droso}s.fastq; done +``` + +Terminal logs for alignment and summary files are included in the ./alignment folder. + +The generated BAM files were then sorted (using the default sorting, by position) and indexed using SAMtools (v0.1.18, included in the TopHat package): + +```bash +for droso in {57..60}; do ./tophat-2.1.1.Linux_x86_64/samtools_0.1.18 sort ./ERR9905${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_ 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 +``` + +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 DEanalysis.R script for details on the analysis. + +Graphs generated by the script include an MA plot: + +![MA plot](/analysis.1412/graphs/MA-plot.png?raw=true "MA plot") + +And a plot of the dispersion estimates: + +![Dispersion estimate plot](/analysis.1412/graphs/DispersionEst-plot.png?raw=true "Dispersion estimate plot") + +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 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 diff --git a/analysis.1412/graphs/DispersionEst-plot.png b/analysis.1412/graphs/DispersionEst-plot.png new file mode 100644 index 0000000..ebd6e8c Binary files /dev/null and b/analysis.1412/graphs/DispersionEst-plot.png differ diff --git a/analysis.1412/graphs/MA-plot.png b/analysis.1412/graphs/MA-plot.png new file mode 100644 index 0000000..f2c67b9 Binary files 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