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Genome Index and Alignment- STAR

Hi...

I'm new to the field of bioinformatics. I'm trying to align RNAseq with STAR, but I'm having problems. I do not know if it's time to generate the genome index or alignment.

I am generating the genome index for Rattus novergicus (ftp://ftp.ensembl.org/pub/release-94/fasta/rattus_norvegicus/dna/). I downloaded the FASTA files (chr1-chr20, chrY, chrX and chr MT) and the GTF file. that was my script:

STAR --runThreadN 8 --runMode genomeGenerate --genomeDir /home/acamar2/files/Rnor_6.0 --genomeFastaFiles /home/acamar2/files/Fasta/Rattus_norvegicus.Rnor_6.0.dna.chromosome.1.fa /home/acamar2/files/Fasta/Rattus_norvegicus.Rnor_6.0.dna.chromosome.2.fa /home/acamar2/files/Fasta/Rattus_norvegicus.Rnor_6.0.dna.chromosome.3.fa /home/acamar2/files/Fasta/Rattus_norvegicus.Rnor_6.0.dna.chromosome.4.fa /home/acamar2/files/Fasta/Rattus_norvegicus.Rnor_6.0.dna.chromosome.5.fa /home/acamar2/files/Fasta/Rattus_norvegicus.Rnor_6.0.dna.chromosome.5.fa /home/acamar2/files/Fasta/Rattus_norvegicus.Rnor_6.0.dna.chromosome.6.fa /home/acamar2/files/Fasta/Rattus_norvegicus.Rnor_6.0.dna.chromosome.8.fa /home/acamar2/files/Fasta/Rattus_norvegicus.Rnor_6.0.dna.chromosome.9.fa /home/acamar2/files/Fasta/Rattus_norvegicus.Rnor_6.0.dna.chromosome.10.fa /home/acamar2/files/Fasta/Rattus_norvegicus.Rnor_6.0.dna.chromosome.11.fa /home/acamar2/files/Fasta/Rattus_norvegicus.Rnor_6.0.dna.chromosome.12.fa /home/acamar2/files/Fasta/Rattus_norvegicus.Rnor_6.0.dna.chromosome.13.fa /home/acamar2/files/Fasta/Rattus_norvegicus.Rnor_6.0.dna.chromosome.14.fa /home/acamar2/files/Fasta/Rattus_norvegicus.Rnor_6.0.dna.chromosome.15.fa /home/acamar2/files/Fasta/Rattus_norvegicus.Rnor_6.0.dna.chromosome.16.fa /home/acamar2/files/Fasta/Rattus_norvegicus.Rnor_6.0.dna.chromosome.17.fa /home/acamar2/files/Fasta/Rattus_norvegicus.Rnor_6.0.dna.chromosome.18.fa /home/acamar2/files/Fasta/Rattus_norvegicus.Rnor_6.0.dna.chromosome.19.fa /home/acamar2/files/Fasta/Rattus_norvegicus.Rnor_6.0.dna.chromosome.20.fa /home/acamar2/files/Fasta/Rattus_norvegicus.Rnor_6.0.dna.chromosome.Y.fa /home/acamar2/files/Fasta/Rattus_norvegicus.Rnor_6.0.dna.chromosome.MT.fa /home/acamar2/files/Fasta/Rattus_norvegicus.Rnor_6.0.dna.chromosome.X.fa --sjdbGTFfile /home/acamar2/files/annotation/Rattus_norvegicus.Rnor_6.0.94.gtf

The generated files were:

drwxr-xr-x 2 acamar2 acamar2 4,0K Nov 28 15:58 .
drwxr-xr-x 5 acamar2 acamar2   50 Nov 28 15:58 ..
-rw-rw-r-- 1 acamar2 acamar2  217 Nov 28 14:56 chrLength.txt
-rw-rw-r-- 1 acamar2 acamar2  275 Nov 28 14:56 chrNameLength.txt
-rw-rw-r-- 1 acamar2 acamar2   58 Nov 28 14:56 chrName.txt
-rw-rw-r-- 1 acamar2 acamar2  245 Nov 28 14:56 chrStart.txt
-rw-rw-r-- 1 acamar2 acamar2  11M Nov 28 15:44 exonGeTrInfo.tab
-rw-rw-r-- 1 acamar2 acamar2 4,5M Nov 28 15:44 exonInfo.tab
-rw-rw-r-- 1 acamar2 acamar2 606K Nov 28 15:44 geneInfo.tab
-rw-rw-r-- 1 acamar2 acamar2 2,7G Nov 28 15:49 Genome
-rw-rw-r-- 1 acamar2 acamar2 4,0K Nov 28 14:55 genomeParameters.txt
-rw-rw-r-- 1 acamar2 acamar2  21G Nov 28 15:53 SA
-rw-rw-r-- 1 acamar2 acamar2 1,5G Nov 28 15:53 SAindex
-rw-rw-r-- 1 acamar2 acamar2 5,6M Nov 28 15:44 sjdbInfo.txt
-rw-rw-r-- 1 acamar2 acamar2 4,4M Nov 28 15:44 sjdbList.fromGTF.out.tab
-rw-rw-r-- 1 acamar2 acamar2 4,4M Nov 28 15:44 sjdbList.out.tab
-rw-rw-r-- 1 acamar2 acamar2 2,4M Nov 28 15:44 transcriptInfo.tab

I followed the alignment of the samples with the following script:

STAR --runThreadN 8 --genomeDir /home/acamar2/files/Rnor_6.0 --sjdbGTFfile /home/acamar2/files/Rnor_6.0/Rattus_norvegicus.Rnor_6.0.94.gtf --readFilesIn /home/acamar2/rawdata/1_1.fastq.gz,/home/acamar2/rawdata/1_2.fastq.gz,/home/acamar2/rawdata/2_1.fastq.gz,/home/acamar2/rawdata/2_2.fastq.gz,/home/acamar2/rawdata/3_1.fastq.gz,/home/acamar2/rawdata/3_2.fastq.gz,/home/acamar2/rawdata/4_1.fastq.gz,/home/acamar2/rawdata/4_2.fastq.gz,/home/acamar2/rawdata/5_1.fastq.gz,/home/acamar2/rawdata/5_2.fastq.gz,/home/acamar2/rawdata/6_1.fastq.gz,/home/acamar2/rawdata/6_2.fastq.gz,/home/acamar2/rawdata/7_1.fastq.gz,/home/acamar2/rawdata/7_2.fastq.gz,/home/acamar2/rawdata/8_1.fastq.gz,/home/acamar2/rawdata/8_2.fastq`.gz --readFilesCommand gunzip --quantMode TranscriptomeSAM GeneCounts --outSAMtype BAM SortedByCoordinate --outFileNamePrefix /home/acamar2/STARoutput/fastq

The generated files were:

drwxr-xr-x 4 acamar2 acamar2 4,0K Nov 28 16:42 .
drwxr-xr-x 7 acamar2 acamar2  115 Nov 28 16:00 ..
-rw-rw-r-- 1 acamar2 acamar2   28 Nov 28 16:42 fastqAligned.sortedByCoord.out.bam
-rw-rw-r-- 1 acamar2 acamar2 567K Nov 28 16:42 fastqAligned.toTranscriptome.out.bam
-rw-rw-r-- 1 acamar2 acamar2 1,8K Nov 28 16:42 fastqLog.final.out
-rw-rw-r-- 1 acamar2 acamar2 461K Nov 28 16:42 fastqLog.out
-rw-r--r-- 1 acamar2 acamar2 268M Nov 19 16:30 Rattus_norvegicus.Rnor_6.0.94.gtf
-rw-rw-r-- 1 acamar2 acamar2  246 Nov 28 16:42 fastqLog.progress.out
-rw-rw-r-- 1 acamar2 acamar2 797K Nov 28 16:42 fastqReadsPerGene.out.tab
-rw-rw-r-- 1 acamar2 acamar2    0 Nov 28 16:42 fastqSJ.out.tab
drwx------ 2 acamar2 acamar2 4,0K Nov 28 16:03 fastq_STARgenome
drwx------ 3 acamar2 acamar2   20 Nov 28 16:42 fastq_STARtmp

But, my files ReadsPerGene and Log.final.out appear zeroed.

N_unmapped  0   0   0
N_multimapping  0   0   0
N_noFeature 0   0   0
N_ambiguous 0   0   0
ENSRNOG00000046319  0   0   0
ENSRNOG00000047964  0   0   0
ENSRNOG00000050370  0   0   0
ENSRNOG00000032365  0   0   0
ENSRNOG00000040300  0   0   0
ENSRNOG00000058808  0   0   0
ENSRNOG00000061316  0   0   0
ENSRNOG00000050129  0   0   0
ENSRNOG00000040316  0   0   0
ENSRNOG00000023659  0   0   0
ENSRNOG00000029897  0   0   0
ENSRNOG00000042852  0   0   0
ENSRNOG00000061806  0   0   0
ENSRNOG00000055877  0   0   0
ENSRNOG00000014303  0   0   0
ENSRNOG00000051899  0   0   0
ENSRNOG00000014330  0   0   0
ENSRNOG00000049505  0   0   0
ENSRNOG00000014916  0   0   0
ENSRNOG00000014996  0   0   0
ENSRNOG00000015239  0   0   0
ENSRNOG00000055508  0   0   0
ENSRNOG00000053710  0   0   0
ENSRNOG00000015552  0   0   0
ENSRNOG00000016041  0   0   0
ENSRNOG00000016054  0   0   0
ENSRNOG00000016381  0   0   0
ENSRNOG00000054005  0   0   0
ENSRNOG00000059137  0   0   0
ENSRNOG00000013160  0   0   0
ENSRNOG00000052788  0   0   0
ENSRNOG00000023549  0   0   0
ENSRNOG00000031859  0   0   0
ENSRNOG00000013351  0   0   0


                Started job on |    Nov 28 17:12:06
                             Started mapping on |   Nov 28 17:15:03
                                    Finished on |   Nov 28 17:51:41
       Mapping speed, Million of reads per hour |   0.00

                          Number of input reads |   0
                      Average input read length |   0
                                    UNIQUE READS:
                   Uniquely mapped reads number |   0
                        Uniquely mapped reads % |   0.00%
                          Average mapped length |   0.00
                       Number of splices: Total |   0
            Number of splices: Annotated (sjdb) |   0
                       Number of splices: GT/AG |   0
                       Number of splices: GC/AG |   0
                       Number of splices: AT/AC |   0
               Number of splices: Non-canonical |   0
                      Mismatch rate per base, % |   -nan%
                         Deletion rate per base |   0.00%
                        Deletion average length |   0.00
                        Insertion rate per base |   0.00%
                       Insertion average length |   0.00
                             MULTI-MAPPING READS:
        Number of reads mapped to multiple loci |   0
             % of reads mapped to multiple loci |   0.00%
        Number of reads mapped to too many loci |   0
             % of reads mapped to too many loci |   0.00%
                                  UNMAPPED READS:
       % of reads unmapped: too many mismatches |   0.00%
                 % of reads unmapped: too short |   0.00%
                     % of reads unmapped: other |   0.00%
                                  CHIMERIC READS:
                       Number of chimeric reads |   0
                            % of chimeric reads |   0.00%

I do not know if I'm generating the genome index or doing the alignment correctly. Can someone help me?

Thanks

rna-seq genome alignment star
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updated by GenoMax GenoMax • written by carolgalah • carolgalah

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written by Kevin Blighe Kevin Blighe

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