+ wget -q -nc http://data.biostarhandbook.com/books/rnaseq/data/golden.genome.tar.gz + tar xzvf golden.genome.tar.gz + wget -q -nc http://data.biostarhandbook.com/books/rnaseq/data/golden.reads.tar.gz + tar zxvf golden.reads.tar.gz + IDX=refs/genome.fa + hisat2-build refs/genome.fa refs/genome.fa Settings: Output files: "refs/genome.fa.*.ht2" 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 Local offset rate: 3 (one in 8) Local fTable chars: 6 Local sequence length: 57344 Local sequence overlap between two consecutive indexes: 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: refs/genome.fa Reading reference sizes Time reading reference sizes: 00:00:00 Calculating joined length Writing header Reserving space for joined string Joining reference sequences Time to join reference sequences: 00:00:00 Time to read SNPs and splice sites: 00:00:00 Using parameters --bmax 24141 --dcv 1024 Doing ahead-of-time memory usage test Passed! Constructing with these parameters: --bmax 24141 --dcv 1024 Constructing suffix-array element generator Converting suffix-array elements to index image Allocating ftab, absorbFtab Entering GFM loop Exited GFM loop fchr[A]: 0 fchr[C]: 47248 fchr[G]: 65274 fchr[T]: 83911 fchr[$]: 128755 Exiting GFM::buildToDisk() Returning from initFromVector Wrote 4237485 bytes to primary GFM file: refs/genome.fa.1.ht2 Wrote 32196 bytes to secondary GFM file: refs/genome.fa.2.ht2 Re-opening _in1 and _in2 as input streams Returning from GFM constructor Returning from initFromVector Wrote 62331 bytes to primary GFM file: refs/genome.fa.5.ht2 Wrote 32708 bytes to secondary GFM file: refs/genome.fa.6.ht2 Re-opening _in5 and _in5 as input streams Returning from HierEbwt constructor Headers: len: 128755 gbwtLen: 128756 nodes: 128756 sz: 32189 gbwtSz: 32190 lineRate: 6 offRate: 4 offMask: 0xfffffff0 ftabChars: 10 eftabLen: 0 eftabSz: 0 ftabLen: 1048577 ftabSz: 4194308 offsLen: 8048 offsSz: 32192 lineSz: 64 sideSz: 64 sideGbwtSz: 48 sideGbwtLen: 192 numSides: 671 numLines: 671 gbwtTotLen: 42944 gbwtTotSz: 42944 reverse: 0 linearFM: Yes Total time for call to driver() for forward index: 00:00:00 + samtools faidx refs/genome.fa + parallel -j 1 echo '{1}_{2}' ::: BORED EXCITED ::: 1 2 3 + mkdir -p bam + cat ids + parallel 'hisat2 -x refs/genome.fa -1 reads/{}_R1.fq -2 reads/{}_R2.fq | samtools sort > bam/{}.bam' 112193 reads; of these: 112193 (100.00%) were paired; of these: 320 (0.29%) aligned concordantly 0 times 111873 (99.71%) aligned concordantly exactly 1 time 0 (0.00%) aligned concordantly >1 times ---- 320 pairs aligned concordantly 0 times; of these: 318 (99.38%) aligned discordantly 1 time ---- 2 pairs aligned 0 times concordantly or discordantly; of these: 4 mates make up the pairs; of these: 0 (0.00%) aligned 0 times 4 (100.00%) aligned exactly 1 time 0 (0.00%) aligned >1 times 100.00% overall alignment rate 123093 reads; of these: 123093 (100.00%) were paired; of these: 371 (0.30%) aligned concordantly 0 times 122696 (99.68%) aligned concordantly exactly 1 time 26 (0.02%) aligned concordantly >1 times ---- 371 pairs aligned concordantly 0 times; of these: 363 (97.84%) aligned discordantly 1 time ---- 8 pairs aligned 0 times concordantly or discordantly; of these: 16 mates make up the pairs; of these: 0 (0.00%) aligned 0 times 14 (87.50%) aligned exactly 1 time 2 (12.50%) aligned >1 times 100.00% overall alignment rate 158009 reads; of these: 158009 (100.00%) were paired; of these: 536 (0.34%) aligned concordantly 0 times 157473 (99.66%) aligned concordantly exactly 1 time 0 (0.00%) aligned concordantly >1 times ---- 536 pairs aligned concordantly 0 times; of these: 515 (96.08%) aligned discordantly 1 time ---- 21 pairs aligned 0 times concordantly or discordantly; of these: 42 mates make up the pairs; of these: 0 (0.00%) aligned 0 times 42 (100.00%) aligned exactly 1 time 0 (0.00%) aligned >1 times 100.00% overall alignment rate 137581 reads; of these: 137581 (100.00%) were paired; of these: 21838 (15.87%) aligned concordantly 0 times 115743 (84.13%) aligned concordantly exactly 1 time 0 (0.00%) aligned concordantly >1 times ---- 21838 pairs aligned concordantly 0 times; of these: 21023 (96.27%) aligned discordantly 1 time ---- 815 pairs aligned 0 times concordantly or discordantly; of these: 1630 mates make up the pairs; of these: 0 (0.00%) aligned 0 times 1630 (100.00%) aligned exactly 1 time 0 (0.00%) aligned >1 times 100.00% overall alignment rate 196673 reads; of these: 196673 (100.00%) were paired; of these: 660 (0.34%) aligned concordantly 0 times 195995 (99.66%) aligned concordantly exactly 1 time 18 (0.01%) aligned concordantly >1 times ---- 660 pairs aligned concordantly 0 times; of these: 644 (97.58%) aligned discordantly 1 time ---- 16 pairs aligned 0 times concordantly or discordantly; of these: 32 mates make up the pairs; of these: 0 (0.00%) aligned 0 times 32 (100.00%) aligned exactly 1 time 0 (0.00%) aligned >1 times 100.00% overall alignment rate 237018 reads; of these: 237018 (100.00%) were paired; of these: 376 (0.16%) aligned concordantly 0 times 236642 (99.84%) aligned concordantly exactly 1 time 0 (0.00%) aligned concordantly >1 times ---- 376 pairs aligned concordantly 0 times; of these: 366 (97.34%) aligned discordantly 1 time ---- 10 pairs aligned 0 times concordantly or discordantly; of these: 20 mates make up the pairs; of these: 0 (0.00%) aligned 0 times 20 (100.00%) aligned exactly 1 time 0 (0.00%) aligned >1 times 100.00% overall alignment rate + cat ids + parallel 'samtools index bam/{}.bam' + cat ids + parallel 'bedtools genomecov -ibam bam/{}.bam -split -bg > bam/{}.bg' + cat ids + parallel 'bedGraphToBigWig bam/{}.bg refs/genome.fa.fai bam/{}.bw' + featureCounts -p -a refs/features.gff -o counts.txt bam/BORED_1.bam bam/BORED_2.bam bam/BORED_3.bam bam/EXCITED_1.bam bam/EXCITED_2.bam bam/EXCITED_3.bam ========== _____ _ _ ____ _____ ______ _____ ===== / ____| | | | _ \| __ \| ____| /\ | __ \ ===== | (___ | | | | |_) | |__) | |__ / \ | | | | ==== \___ \| | | | _ <| _ /| __| / /\ \ | | | | ==== ____) | |__| | |_) | | \ \| |____ / ____ \| |__| | ========== |_____/ \____/|____/|_| \_\______/_/ \_\_____/ v1.6.4 //========================== featureCounts setting ===========================\\ || || || Input files : 6 BAM files || || P BORED_1.bam || || P BORED_2.bam || || P BORED_3.bam || || P EXCITED_1.bam || || P EXCITED_2.bam || || P EXCITED_3.bam || || || || Output file : counts.txt || || Summary : counts.txt.summary || || Annotation : features.gff (GTF) || || Dir for temp files : ./ || || || || Threads : 1 || || Level : meta-feature level || || Paired-end : yes || || Multimapping reads : not counted || || Multi-overlapping reads : not counted || || Min overlapping bases : 1 || || || || Chimeric reads : counted || || Both ends mapped : not required || || || \\============================================================================// //================================= Running ==================================\\ || || || Load annotation file features.gff ... || || Features : 92 || || Meta-features : 92 || || Chromosomes/contigs : 1 || || || || Process BAM file BORED_1.bam... || || Paired-end reads are included. || || Assign alignments (paired-end) to features... || || || || WARNING: reads from the same pair were found not adjacent to each || || other in the input (due to read sorting by location or || || reporting of multi-mapping read pairs). || || || || Pairing up the read pairs. || || || || Total alignments : 112193 || || Successfully assigned alignments : 112193 (100.0%) || || Running time : 0.00 minutes || || || || Process BAM file BORED_2.bam... || || Paired-end reads are included. || || Assign alignments (paired-end) to features... || || || || WARNING: reads from the same pair were found not adjacent to each || || other in the input (due to read sorting by location or || || reporting of multi-mapping read pairs). || || || || Pairing up the read pairs. || || || || Total alignments : 182196 || || Successfully assigned alignments : 182193 (100.0%) || || Running time : 0.01 minutes || || || || Process BAM file BORED_3.bam... || || Paired-end reads are included. || || Assign alignments (paired-end) to features... || || || || WARNING: reads from the same pair were found not adjacent to each || || other in the input (due to read sorting by location or || || reporting of multi-mapping read pairs). || || || || Pairing up the read pairs. || || || || Total alignments : 123121 || || Successfully assigned alignments : 123067 (100.0%) || || Running time : 0.00 minutes || || || || Process BAM file EXCITED_1.bam... || || Paired-end reads are included. || || Assign alignments (paired-end) to features... || || || || WARNING: reads from the same pair were found not adjacent to each || || other in the input (due to read sorting by location or || || reporting of multi-mapping read pairs). || || || || Pairing up the read pairs. || || || || Total alignments : 237018 || || Successfully assigned alignments : 237014 (100.0%) || || Running time : 0.01 minutes || || || || Process BAM file EXCITED_2.bam... || || Paired-end reads are included. || || Assign alignments (paired-end) to features... || || || || WARNING: reads from the same pair were found not adjacent to each || || other in the input (due to read sorting by location or || || reporting of multi-mapping read pairs). || || || || Pairing up the read pairs. || || || || Total alignments : 158009 || || Successfully assigned alignments : 158009 (100.0%) || || Running time : 0.01 minutes || || || || Process BAM file EXCITED_3.bam... || || Paired-end reads are included. || || Assign alignments (paired-end) to features... || || || || WARNING: reads from the same pair were found not adjacent to each || || other in the input (due to read sorting by location or || || reporting of multi-mapping read pairs). || || || || Pairing up the read pairs. || || || || Total alignments : 196691 || || Successfully assigned alignments : 196653 (100.0%) || || Running time : 0.01 minutes || || || || || || Summary of counting results can be found in file "counts.txt.summary" || || || \\============================================================================// + curl http://data.biostarhandbook.com/books/rnaseq/code/edger.r % Total % Received % Xferd Average Speed Time Time Time Current Dload Upload Total Spent Left Speed 0 0 0 0 0 0 0 0 --:--:-- --:--:-- --:--:-- 0 100 4148 100 4148 0 0 25447 0 --:--:-- --:--:-- --:--:-- 25447 + curl http://data.biostarhandbook.com/books/rnaseq/code/heatmap.r % Total % Received % Xferd Average Speed Time Time Time Current Dload Upload Total Spent Left Speed 0 0 0 0 0 0 0 0 --:--:-- --:--:-- --:--:-- 0 100 1874 100 1874 0 0 11154 0 --:--:-- --:--:-- --:--:-- 11154 + cat counts.txt + Rscript edger.r 3x3 + cat results.csv + Rscript heatmap.r Warning message: package ‘gplots’ was built under R version 3.6.3