DEXSeq gives most of the reads as "_empty"

Question

[rohitsatyam102]

Hi All!

I am new to DEXSeq. When looking at the counts' files, I see a lot of reads are _empty assignment. From this thread, I learned these are the reads DEXSeq doesn't consider in the actual counts and are ones that fall in the intronic region. Since I am bound to use "-r no" option (because the gene I am interested in a gene that gets aggregated with 5 neighbor genes which are undesirable for my analysis), I see read assigned to _empty .

ENSG00000288588.1:002   0
ENSG00000288588.1:003   0
ENSG00000288588.1:004   0
ENSG00000288588.1:005   0
ENSG00000288588.1:006   0
_ambiguous      26889
_ambiguous_readpair_position    0
_empty  16171092
_lowaqual       0
_notaligned     0

Using '-r no' option

ENSG00000288588.1:002   0
ENSG00000288588.1:003   0
ENSG00000288588.1:004   0
ENSG00000288588.1:005   0
ENSG00000288588.1:006   0
_ambiguous      37555
_ambiguous_readpair_position    0
_empty  20575375
_lowaqual       0
_notaligned     0

My data contains ~63 million reads that are uniquely mappable and rest ~22 million are multi-mappable reads giving ~85 million mappable reads. Loosing 16-20 million reads means losing 1/4th of the data.

Relevant Codes

python ./DEXSeq/python_scripts/dexseq_prepare_annotation.py -r no /rnaseq/gencode.v33.annotation.gtf gencode.v33.gff 2> gff.stderr

python ./DEXSeq/python_scripts/dexseq_count.py -p yes -r pos -s reverse -a 10 gencode.v33.gff $p ${name}.Readcounts.txt 2> ${name}.stderr

[rohitsatyam102]

Alignments were performed using RSEM, GRCh38, v33 primary assembly, STAR aligner.

I used the following command

bsub -e rsem.e -o rsem.o -n 32 -q regularq rsem-calculate-expression --star-output-genome-bam --strandedness reverse -p 32 --calc-pme --calc-ci --keep-intermediate-files --append-names --sort-bam-by-coordinate --paired-end --estimate-rspd --star --star-path /anaconda3/bin/ ./ananda_rnaseq/trimmomatic/paired/Contr1_S15_L004_R1_p.fastq ./ananda_rnaseq/trimmomatic/paired/Contr1_S15_L004_R2_p.fastq ./archive/rnaseq/rsem ./scratch/last_rsem_test/Contr1_S15_L004 2> ./scratch/last_rsem_test/Contr1_S15_L004.stderr