[neokao]

I used homebrew to install gcc again. The installed path is /usr/local/Cellar/gcc49.

Then I did make STARforMacStatic CXX=/usr/local/Cellar/gcc49/bin/g++-4.9 and I got another error:
make: *** No rule to make target `STARforMacStatic'. Stop.

Really frustrating now.

[alexdobin]

I used homebrew to install gcc again. The installed path is /usr/local/Cellar/gcc49.

Then I did make STARforMacStatic CXX=/usr/local/Cellar/gcc49/bin/g++-4.9 and I got another error:
make: *** No rule to make target `STARforMacStatic'. Stop.

Really frustrating now.

Are you running make from the STAR source directory? This error often occurs if make is run from a wrong directory.
If you do:
grep STARforMacStatic Makefile
it will show you the STARforMacStatic target in the Makefile:
STARforMacStatic : CCFLAGS=-D'COMPILE_FOR_MAC' -I ./Mac_Include/ $(CCFLAGS_main)
STARforMacStatic : parametersDefault.xxd $(OBJECTS)


Cheers
Alex

[neokao]

i switched to source directory.
grep STARforMacStatic Makefile
STARforMacStatic : CCFLAGS=-D'COMPILE_FOR_MAC' -I ./Mac_Include/ $(CCFLAGS_main)
STARforMacStatic : parametersDefault.xxd $(OBJECTS)

Then I got this error:

make STARforMacStatic CXX=/usr/local/Cellar/gcc49/bin/g++-4.9
/usr/local/Cellar/gcc49/bin/g++-4.9 -o STAR -D'COMPILE_FOR_MAC' -I ./Mac_Include/ -O3 -pipe -std=c++0x -Wall -Wextra -fopenmp -D'COMPILATION_TIME_PLACE="Wed Apr 8 12:34:50 CST 2015 :/Users/Matrix_Macmini/NGS/STAR-STAR_2.4.0k/source"' -pthread -lz -static-libgcc htslib/libhts.a PackedArray.o SuffixArrayFuns.o STAR.o Parameters.o InOutStreams.o SequenceFuns.o Genome.o Stats.o Transcript.o Transcript_alignScore.o ReadAlign.o ReadAlign_storeAligns.o ReadAlign_stitchPieces.o ReadAlign_multMapSelect.o ReadAlign_mapOneRead.o readLoad.o ReadAlignChunk.o ReadAlignChunk_processChunks.o ReadAlignChunk_mapChunk.o OutSJ.o outputSJ.o blocksOverlap.o ThreadControl.o sysRemoveDir.o ReadAlign_maxMappableLength2strands.o binarySearch2.o ReadAlign_outputAlignments.o ReadAlign_outputTranscriptSAM.o ReadAlign_outputTranscriptSJ.o ReadAlign_outputTranscriptCIGARp.o ReadAlign_createExtendWindowsWithAlign.o ReadAlign_assignAlignToWindow.o ReadAlign_oneRead.o ReadAlign_stitchWindowSeeds.o ReadAlign_chimericDetection.o stitchWindowAligns.o extendAlign.o stitchAlignToTranscript.o alignSmithWaterman.o genomeGenerate.o TimeFunctions.o ErrorWarning.o loadGTF.o streamFuns.o stringSubstituteAll.o Transcriptome.o Transcriptome_quantAlign.o ReadAlign_quantTranscriptome.o Quantifications.o Transcriptome_geneCountsAddAlign.o sjdbLoadFromFiles.o sjdbLoadFromStream.o sjdbPrepare.o sjdbBuildIndex.o mapThreadsSpawn.o Parameters_openReadsFiles.cpp Parameters_closeReadsFiles.cpp BAMoutput.o BAMfunctions.o ReadAlign_alignBAM.o BAMbinSortByCoordinate.o signalFromBAM.o bamRemoveDuplicates.o BAMbinSortUnmapped.o bam_cat.o
make: /usr/local/Cellar/gcc49/bin/g++-4.9: No such file or directory
make: *** [STARforMacStatic] Error 1

[alexdobin]

i switched to source directory.
grep STARforMacStatic Makefile
STARforMacStatic : CCFLAGS=-D'COMPILE_FOR_MAC' -I ./Mac_Include/ $(CCFLAGS_main)
STARforMacStatic : parametersDefault.xxd $(OBJECTS)

Then I got this error:

make STARforMacStatic CXX=/usr/local/Cellar/gcc49/bin/g++-4.9
/usr/local/Cellar/gcc49/bin/g++-4.9 -o STAR -D'COMPILE_FOR_MAC' -I ./Mac_Include/ -O3 -pipe -std=c++0x -Wall -Wextra -fopenmp -D'COMPILATION_TIME_PLACE="Wed Apr 8 12:34:50 CST 2015 :/Users/Matrix_Macmini/NGS/STAR-STAR_2.4.0k/source"' -pthread -lz -static-libgcc htslib/libhts.a PackedArray.o SuffixArrayFuns.o STAR.o Parameters.o InOutStreams.o SequenceFuns.o Genome.o ...
make: /usr/local/Cellar/gcc49/bin/g++-4.9: No such file or directory
make: *** [STARforMacStatic] Error 1

It looks like you path to /usr/local/Cellar/gcc49/bin/g++-4.9 is broken. Can you run /usr/local/Cellar/gcc49/bin/g++-4.9 -v ?

I have posted the 2.4.0k Mac executable here:
http://labshare.cshl.edu/shares/ging...SX_x86_64/STAR
Hopefully, it will work on your system and save you the compiling troubles.

Cheers
Alex

[neokao]

I did : /usr/local/Cellar/gcc49/bin/g++-4.9 -v and got this:
-bash: /usr/local/Cellar/gcc49/bin/g++-4.9: No such file or directory.

Anyway, I downloaded your 2.4.0k Mac executable and it worked fine with that particular .fq file now. Thanks a lot for all the patience helping me.

[IonTom]

Dear Alex,

i started to use STAR and am really happy with it.

There are just a few questions:

1. We have different settings. In one we have 150 bases single end reads
in the other 75 bases paired end reads. I am using a gtf file with annotated splice junctions. What would be the ideal value for --sjdbOverhang in the single end case be (149 ?) or would the general value of 100 should not be fine and how does read trimming influence this choice ? As then the read lengths would vary.
Why does it even make a difference if i have a read length of 150 and an overhang of 150 there would be no splicing anyway. So the values for --sjdbOverhang ould just have to be higher than the maximum read length ?

2. When using a gtf annotation and i use the filter BySJout option
does it remove all reads that that contain non annotated splices. Because in the log file it says so.

3. Is there a best practice way to filter unreliable novel junctions before the two step procedure.
And does the two step procedure hav an influence on differential expression for well annotated genomes
such as arabidopsis.

4. We plan to have many measurements over a long time, what would be the
ideal setting of STAR to make sure that the values are comparable for differential expression.

5. What happens when only one read of a read was mapped are both discarded ?
Do both reads always have the same mapping quality ?



May thanks for your help.

[alexdobin]

Dear Alex,

i started to use STAR and am really happy with it.

There are just a few questions:

1. We have different settings. In one we have 150 bases single end reads
in the other 75 bases paired end reads. I am using a gtf file with annotated splice junctions. What would be the ideal value for --sjdbOverhang in the single end case be (149 ?) or would the general value of 100 should not be fine and how does read trimming influence this choice ? As then the read lengths would vary.
Why does it even make a difference if i have a read length of 150 and an overhang of 150 there would be no splicing anyway. So the values for --sjdbOverhang ould just have to be higher than the maximum read length ?

2. When using a gtf annotation and i use the filter BySJout option
does it remove all reads that that contain non annotated splices. Because in the log file it says so.

3. Is there a best practice way to filter unreliable novel junctions before the two step procedure.
And does the two step procedure hav an influence on differential expression for well annotated genomes
such as arabidopsis.

4. We plan to have many measurements over a long time, what would be the
ideal setting of STAR to make sure that the values are comparable for differential expression.

5. What happens when only one read of a read was mapped are both discarded ?
Do both reads always have the same mapping quality ?

May thanks for your help.

Hi @IonTom,

good questions, here are my answers:

1. The --sjdbOvehang=100 (which is set by default in the latest versions of STAR) is fine in most of the applications. Users can still fine tune it, with impact being higher for short reads (<50b). These thread , thread discuss the workings of --sjdbOverhang in more detail.

2. --outFilterType BySJout option will keep reads splicing across the annotated junctions, as well as novel junctions that pass filtering by --outSJfilter* options (only those filtered junctions that are output in SJ.out.tab). In other words, it synchronizes the alignments (SAM or BAM) with the SJ.out.tab file. --outSJfilter* filters include filtering by the minimum splice ovehang, number of reads per junction, intron length, and closeness to other junctions.

3. Since the junctions for the 2-pass mapping are taken from the SJ.out.tab file, they are already filtered with some default values of the --outSJfilter* filters. You can review this filtering to see if you want to make it more or less stringent. It is advisable to remove junctions from those references that are not supposed to splice, such as chrM or spike-ins, these are false-positives and may slow the 2nd pass mapping.
Also, if you get a large number of non-canonical junctions, it may be helpful to filter them more stringently.

4. To keep things comparable over long time on the pipeline side, you need to keep the same run parameters, genome, and annotations. STAR versions/patches typically produce exactly the same results, unless specific changes in the mapping algorithm are announced.

5. By default, only correctly paired alignments are output. It is possible (but not recommended) to output single-end or non-concordant pairs.
The mapping quality of two reads in a pair is not required to be the same, on the contrary, the mapping matrics of the whole pair (alignment score, length, number of mismatches, etc) are controlled.

Cheers
Alex

[coryfunk]

Alex,

I'ved used older versions of STAR before and I'm now trying to use the latest 2.4.2a. I'd like to try the --quantMode GeneCounts option and I'm having trouble. From the documentation, as long as I included the -sjdbGTFfile option in building my indices, I should be able to use the --quantMode option. Here's how I built my indices:

STAR --runMode genomeGenerate --genomeDir /mnt/indices --genomeFastaFiles /mnt/genome/Homo_sapiens.GRCh38.dna.SORTED.fa --sjdbGTFfile /mnt/transcriptome/Homo_sapiens.GRCh38.77.gtf --runThreadN 32

Here's how I'm trying to run STAR:

STAR --genomeDir /mnt/indices --readFilesIn /mnt/data/NA10-080.FCC6178ACXX_L3_R1_ITAGCTT.fastq.gz /mnt/data/NA10-080.FCC6178ACXX_L3_R2_ITAGCTT.fastq.gz --readFilesCommand zcat -sjdbGTFfile /mnt/transcriptome/Homo_sapiens.GRCh38.77.gtf --quantMode TranscriptomeSAM GeneCounts --runThreadN 32 --outFilterMismatchNmax 8 --outFileNamePrefix NA10-080.star_new --outFilterMultimapNmax 10

I've tried changing the order of some things, but I keep getting the following error:

gzip: invalid option -- 's'
Try `gzip --help' for more information.
Jul 07 19:17:22 ..... Started STAR run
Jul 07 19:17:22 ..... Loading genome
gzip: invalid option -- 's'
Try `gzip --help' for more information.

ERROR_011001: exiting because of *INPUT FILE* error: could not open input file /mnt/indices/transcriptInfo.tab
Solution: check that the file exists and you have read permission for this file
SOLUTION: utilize --sjdbGTFfile /path/to/annotantions.gtf option at the genome generation step or mapping step


At one point it looked like it wasn't correctly pointing to the transcriptInfo.tab file because of how I specified this: --genomeDir /mnt/indices/ giving me the following error:

ERROR_011001: exiting because of *INPUT FILE* error: could not open input file /mnt/indices//transcriptInfo.tab
Solution: check that the file exists and you have read permission for this file
SOLUTION: utilize --sjdbGTFfile /path/to/annotantions.gtf option at the genome generation step or mapping step

I took off the last "/" and it appeared to alter how it assembled the path (see first error message above) but still doesn't seem to find the file.

Thoughts?

[coryfunk]

I think I figured it out.

The indices were created with an older version of STAR, such that when I tried running the newest version of STAR on the old indices, nothing worked.

[profkaramba]

Hi guys

I'm trying to map unmapped human reads to possible contaminants (viruses and bacteria). I download all the complete genomes in ncbi. I have a "concatenated genome" of 9.9Gb in fasta format.

When I try to generate the star index the program stops in

Writing genome to disk... done.
Number of SA indices: 19597668060
SA size in bytes: 85739797763
Jul 29 17:21:10 ... starting to sort  Suffix Array. This may take a long time...
Number of chunks: 43;   chunks size limit: 3994984080 bytes
Jul 29 17:22:26 ... sorting Suffix Array chunks and saving them to disk...
Jul 29 18:59:26 ... loading chunks from disk, packing SA...

no error is reported, it just stops. What can it be?
I tried running it with 6 CPUS and 64Gb of RAM

[profkaramba]

Hi guys

I'm trying to map unmapped human reads to possible contaminants (viruses and bacteria). I download all the complete genomes in ncbi. I have a "concatenated genome" of 9.9Gb in fasta format.

When I try to generate the star index the program stops in

Writing genome to disk... done.
Number of SA indices: 19597668060
SA size in bytes: 85739797763
Jul 29 17:21:10 ... starting to sort  Suffix Array. This may take a long time...
Number of chunks: 43;   chunks size limit: 3994984080 bytes
Jul 29 17:22:26 ... sorting Suffix Array chunks and saving them to disk...
Jul 29 18:59:26 ... loading chunks from disk, packing SA...

no error is reported, it just stops. What can it be?
I tried running it with 6 CPUS and 64Gb of RAM

Ok, problem solved with 200Gb of RAM
Anyway, in the previous run the program didn't report any error. it just stopped working as it had finished... a memory related error should have been given I think!

let's see if I can map now

[GenoMax]

let's see if I can map now

Don't go back to the 64G machine

[stu]

I'm having some difficulty in generating indices with STAR. I'm using a C. elegans WormBase genome and annotations. Every time I attempt to generate indices, I receive the following error, or a variation therin:

SLURM_NODELIST:cnode248
Aug 12 13:54:15 ..... Started STAR run
Aug 12 13:54:15 ... Starting to generate Genome files
Aug 12 13:54:18 ... starting to sort Suffix Array. This may take a long time...
Aug 12 13:54:19 ... sorting Suffix Array chunks and saving them to disk...
Aug 12 13:54:28 ... loading chunks from disk, packing SA...
Aug 12 13:54:31 ... Finished generating suffix array
Aug 12 13:54:31 ... starting to generate Suffix Array index...
Aug 12 13:55:08 ..... Processing annotations GTF
terminate called after throwing an instance of 'std:ut_of_range'
what(): vector::_M_range_check
/tmp/slurmd/job501176/slurm_script: line 5: 14245 Aborted STAR --runThreadN 16 --runMode genomeGenerate --genomeDir ~/CeO2/genomic_ref/ref/ --genomeFastaFiles ~/CeO2/genomic_ref/ref/c_elegans.PRJNA13758.WS240.genomic.fa --sjdbGTFfile ~/CeO2/genomic_ref/ref/c_elegans.PRJNA13758.WS240.annotations.gff

I've made sure that the chromosome names in the FASTA file match the gff. I'm really not sure what else could be wrong.

[GenoMax]

Looks like you are running this under slurm. How much memory are you allocating to this job?

[stu]

I'm pretty sure I'm using 64GB, which is the amount of RAM/node in my cluster. Course, this is an assumption, and we all know what 'assume' does.