Very slow alignment with mix microbial RNA-seq data

[sturkarslan]

Hello,

I have successfully used STAR in the past for analysis of both archaeal and bacterial RNA-seq data. I am currently trying to analyze RNA-seq data that contains RNA mixture from both bacteria and archaea. If i align RNA-seq data to bacteria only, everything works fine however if I perform alignment to archaea, STAR is stuck at " Running STAR alignment" stage without any errors or warnings.

Based on the initial alignment to bacteria, i can see that about 40% of the reads are bacterial origin. Reading other discussions here, i tried to combine GFF and Fasta files for both organisms to create a combined genome. When i try alignment to this genome, STAR will still stall at the same stage.

Any suggestions?

Thank you very much

Serdar

The last message in the log says: 

-----------------------------------------------

Number of real (reference) chromosmes= 3

1       plasmid     202301  0

2       Chromosome      3570858 262144

3       BX950229        1661137 3932160

alignIntronMax=alignMatesGapMax=0, the max intron size will be approximately determined by (2^winBinNbits)*winAnchorDistNbins=589824

Loaded transcript database, nTr=5543

Loaded exon database, nEx=5543

Opening the file: results-combined/01_L1_WT-2-32330323/01-L1-WT-2_star__STARtmp//Unmapped.out.mate1.thread0 ... ok

Opening the file: results-combined/01_L1_WT-2-32330323/01-L1-WT-2_star__STARtmp//Unmapped.out.mate2.thread0 ... ok

Opening the file: results-combined/01_L1_WT-2-32330323/01-L1-WT-2_star__STARtmp//Unmapped.out.mate1.thread1 ... ok

Opening the file: results-combined/01_L1_WT-2-32330323/01-L1-WT-2_star__STARtmp//Unmapped.out.mate2.thread1 ... ok

Opening the file: results-combined/01_L1_WT-2-32330323/01-L1-WT-2_star__STARtmp//Unmapped.out.mate1.thread2 ... ok

Opening the file: results-combined/01_L1_WT-2-32330323/01-L1-WT-2_star__STARtmp//Unmapped.out.mate2.thread2 ... ok

Opening the file: results-combined/01_L1_WT-2-32330323/01-L1-WT-2_star__STARtmp//Unmapped.out.mate1.thread3 ... ok

Opening the file: results-combined/01_L1_WT-2-32330323/01-L1-WT-2_star__STARtmp//Unmapped.out.mate2.thread3 ... ok

Created thread # 1

Created thread # 2

Created thread # 3

Starting to map file # 0

mate 1:   rnaseq-27882859/01_L1_WT-2-32330323/trimmed/01-L1-WT-2_S1_L001_R1_001_paired_trimmed.fastq.gz

mate 2:   rnaseq-27882859/01_L1_WT-2-32330323/trimmed/01-L1-WT-2_S1_L001_R2_001_paired_trimmed.fastq.gz

[Alexander Dobin]

H Serdar,

could you please post the Log.out files of the genome generation and mapping?

Cheers

Alex

[sturkarslan]

Hi Alex,

Thank you for reply. I have done some more troubleshooting on this issue. First of all i was able to run star after combining both genomes. I was able to do that when i used a GTF file instead of GFF. However, alignment to only archaea is still not working with both GFF and GTF.

Here are my Log.out and _star_Log.out files.

Thank you very much.

Serdar

[Alexander Dobin]

Hi Serdar,

there is nothing suspicious in the Log.out files.

Could you please try to run the latest patch of STAR to see if the problem might have been fixed.

If this does not help, please send the link to the genome and a minimal set of reads that reproduces the error.

Cheers

Alex

[sturkarslan]

Hi Alex,

I wasnt able to use the latest STAR due to gcc version issues but i was able to make it work by not providing the gtf file at the genome generation stage but during the STAR alignment. That solved my problem and run is completed.

Thank you very much for all your help

Best

Serdar