Hi,

I got two error messages while running DEXSeq_count.py

"Current read position is smaller than previous reads, is your alignment file properly sorted by position?"

"A chromosome that had finished to be processed before was found again in the alignment file, is your alignment file properly sorted by position?"

These are the codes I used.

For dexseq_prepare_annotation, I used human gtf from ensembl.

python dexseq_prepare_annotation.py /projects/b1036/REFERENCE/Homo_sapiens.GRCh38.84.gtf /projects/b1036/wung/Homo_sapiens.GRCh38.84.DEXSeq.gff

Then I generated sorted SAM files for the samples using STAR Aligner. For example, 

STAR --runThreadN 22 --genomeDir /some/output/dir --readFilesCommand zcat --readFilesIn 15_004_R1.fastq.gz 15_004_R2.fastq.gz --outFileNamePrefix $OUTPUTDIR/15_004_sorted_ --outSAMtype SAM SortedByCoordinate

I repeated these steps for 6 RNA samples: 9_004, 11_006, 15_004, 10_005, 12_012, 16_005

Then I finally ran dexseq_count.py for these 6 samples, using the gff file created from dexseq_prepared_annotation.py and the SAM file generated from STAR aligner

python dexseq_count.py  Homo_sapiens.GRCh38.84.gtf -p yes -r pos 15_004_sorted_Aligned.out.sam 15_004_count.txt

And similarly for other samples (9_004, 11_006, ...)

However, I get error messages 

"Current read position is smaller than previous reads, is your alignment file properly sorted by position?"

"A chromosome that had finished to be processed before was found again in the alignment file, is your alignment file properly sorted by position?"

At first I thought this meant the SAM files are not sorted, but I specified output file type to be SAM file sorted by coordinate when running STAR aligner, so I am lost. 

Are there any mistakes in the steps I took?

Thank you so much in advance,

Wung Jae Lee