Applied Bioinformatics Course Recipes
Results for Alignment based RNA-Seq
Completed
•
Runtime 27 seconds
•
updated 3.4 years ago
by
Istvan Albert
Results generated by running the recipe.
File List
Files created by the recipe run:
bam/BORED_1.bam
12.4 MB
bam/BORED_1.bam.bai
352 bytes
bam/BORED_1.bg
384.4 KB
bam/BORED_1.bw
144.0 KB
bam/BORED_2.bam
15.6 MB
bam/BORED_2.bam.bai
352 bytes
bam/BORED_2.bg
414.2 KB
bam/BORED_2.bw
151.7 KB
bam/BORED_3.bam
13.6 MB
bam/BORED_3.bam.bai
320 bytes
bam/BORED_3.bg
398.3 KB
bam/BORED_3.bw
148.1 KB
bam/EXCITED_1.bam
29.3 MB
bam/EXCITED_1.bam.bai
352 bytes
bam/EXCITED_1.bg
500.9 KB
bam/EXCITED_1.bw
171.4 KB
bam/EXCITED_2.bam
19.7 MB
bam/EXCITED_2.bam.bai
352 bytes
bam/EXCITED_2.bg
440.6 KB
bam/EXCITED_2.bw
158.7 KB
bam/EXCITED_3.bam
24.4 MB
bam/EXCITED_3.bam.bai
400 bytes
bam/EXCITED_3.bg
474.0 KB
bam/EXCITED_3.bw
166.0 KB
counts.txt
5.7 KB
counts.txt.summary
544 bytes
edger.r
4.1 KB
golden.genome.tar.gz
56.1 KB
golden.reads.tar.gz
114.6 MB
heatmap.r
1.8 KB
ids
54 bytes
reads/BORED_1_R1.fq
26.1 MB
reads/BORED_1_R2.fq
26.1 MB
reads/BORED_2_R1.fq
31.9 MB
reads/BORED_2_R2.fq
31.9 MB
reads/BORED_3_R1.fq
28.6 MB
reads/BORED_3_R2.fq
28.6 MB
reads/EXCITED_1_R1.fq
55.0 MB
reads/EXCITED_1_R2.fq
55.0 MB
reads/EXCITED_2_R1.fq
36.7 MB
reads/EXCITED_2_R2.fq
36.7 MB
reads/EXCITED_3_R1.fq
45.7 MB
reads/EXCITED_3_R2.fq
45.7 MB
recipe.sh
1.7 KB
refs/features.gff
12.2 KB
refs/genome.fa
127.4 KB
refs/genome.fa.1.ht2
4.0 MB
refs/genome.fa.2.ht2
31.4 KB
refs/genome.fa.3.ht2
26 bytes
refs/genome.fa.4.ht2
31.4 KB
refs/genome.fa.5.ht2
60.9 KB
refs/genome.fa.6.ht2
31.9 KB
refs/genome.fa.7.ht2
12 bytes
refs/genome.fa.8.ht2
8 bytes
refs/genome.fa.fai
23 bytes
refs/transcripts.fa
83.4 KB
results.csv
7.9 KB
results.pdf
12.4 KB
runlog/input.json
581 bytes
runlog/stderr.txt
17.4 KB
runlog/stdout.txt
4.2 KB
Output Messages
Messages printed to the standard output stream:
refs/genome.fa refs/features.gff refs/transcripts.fa reads/BORED_1_R1.fq reads/BORED_1_R2.fq reads/BORED_2_R1.fq reads/BORED_2_R2.fq reads/BORED_3_R1.fq reads/BORED_3_R2.fq reads/EXCITED_1_R1.fq reads/EXCITED_1_R2.fq reads/EXCITED_2_R1.fq reads/EXCITED_2_R2.fq reads/EXCITED_3_R1.fq reads/EXCITED_3_R2.fq Building DifferenceCoverSample Building sPrime Building sPrimeOrder V-Sorting samples V-Sorting samples time: 00:00:00 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: 00:00:00 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 Split 1, merged 6; iterating... Splitting and merging Splitting and merging time: 00:00:00 Avg bucket size: 18392.7 (target: 24140) Getting block 1 of 7 Reserving size (24141) for bucket 1 Calculating Z arrays for bucket 1 Entering block accumulator loop for bucket 1: bucket 1: 10% bucket 1: 20% bucket 1: 30% bucket 1: 40% bucket 1: 50% bucket 1: 60% bucket 1: 70% bucket 1: 80% bucket 1: 90% bucket 1: 100% Sorting block of length 11794 for bucket 1 (Using difference cover) Sorting block time: 00:00:00 Returning block of 11795 for bucket 1 Getting block 2 of 7 Reserving size (24141) for bucket 2 Calculating Z arrays for bucket 2 Entering block accumulator loop for bucket 2: bucket 2: 10% bucket 2: 20% bucket 2: 30% bucket 2: 40% bucket 2: 50% bucket 2: 60% bucket 2: 70% bucket 2: 80% bucket 2: 90% bucket 2: 100% Sorting block of length 15630 for bucket 2 (Using difference cover) Sorting block time: 00:00:00 Returning block of 15631 for bucket 2 Getting block 3 of 7 Reserving size (24141) for bucket 3 Calculating Z arrays for bucket 3 Entering block accumulator loop for bucket 3: bucket 3: 10% bucket 3: 20% bucket 3: 30% bucket 3: 40% bucket 3: 50% bucket 3: 60% bucket 3: 70% bucket 3: 80% bucket 3: 90% bucket 3: 100% Sorting block of length 19642 for bucket 3 (Using difference cover) Sorting block time: 00:00:00 Returning block of 19643 for bucket 3 Getting block 4 of 7 Reserving size (24141) for bucket 4 Calculating Z arrays for bucket 4 Entering block accumulator loop for bucket 4: bucket 4: 10% bucket 4: 20% bucket 4: 30% bucket 4: 40% bucket 4: 50% bucket 4: 60% bucket 4: 70% bucket 4: 80% bucket 4: 90% bucket 4: 100% Sorting block of length 19091 for bucket 4 (Using difference cover) Sorting block time: 00:00:00 Returning block of 19092 for bucket 4 Getting block 5 of 7 Reserving size (24141) for bucket 5 Calculating Z arrays for bucket 5 Entering block accumulator loop for bucket 5: bucket 5: 10% bucket 5: 20% bucket 5: 30% bucket 5: 40% bucket 5: 50% bucket 5: 60% bucket 5: 70% bucket 5: 80% bucket 5: 90% bucket 5: 100% Sorting block of length 21618 for bucket 5 (Using difference cover) Sorting block time: 00:00:00 Returning block of 21619 for bucket 5 Getting block 6 of 7 Reserving size (24141) for bucket 6 Calculating Z arrays for bucket 6 Entering block accumulator loop for bucket 6: bucket 6: 10% bucket 6: 20% bucket 6: 30% bucket 6: 40% bucket 6: 50% bucket 6: 60% bucket 6: 70% bucket 6: 80% bucket 6: 90% bucket 6: 100% Sorting block of length 22719 for bucket 6 (Using difference cover) Sorting block time: 00:00:00 Returning block of 22720 for bucket 6 Getting block 7 of 7 Reserving size (24141) for bucket 7 Calculating Z arrays for bucket 7 Entering block accumulator loop for bucket 7: bucket 7: 10% bucket 7: 20% bucket 7: 30% bucket 7: 40% bucket 7: 50% bucket 7: 60% bucket 7: 70% bucket 7: 80% bucket 7: 90% bucket 7: 100% Sorting block of length 18255 for bucket 7 (Using difference cover) Sorting block time: 00:00:00 Returning block of 18256 for bucket 7
Other Messages
Messages printed to the standard error stream:
+ 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
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