cannot insert junctions on the fly because of strand GstrandBit problem
yujung-c opened this issue · comments
Hi STAR team! I was trying to use Star for alignment. The output is going to use in rsem-calculate-expression.
When I used star alignment tool, it showed below messages.
【EXITING because of FATAL ERROR: cannot insert sequence on the fly because of strand GstrandBit problem
SOLUTION: please contact STAR author at https://groups.google.com/forum/#!forum/rna-star】
I found the similar history question and tried to set --limitSjdbInsertNsj as 4,000,000, but it doesn't work.
The STAR version was STAR_v2.7.10a
The STAR parameters I used for index generation:
STAR \
--runMode genomeGenerate \
--genomeDir $wd \
--genomeFastaFiles $wd/GRCh38.p14.genome.fa \
--sjdbGTFfile $wd/gencode.v45.annotation.gtf \
--limitSjdbInsertNsj 4000000 \
--runThreadN 20
The processed was finished successfully in Log.out
Then, I ran for STAR alignment.
The STAR parameters I used:
(ps. For looping worked well in --quantMode GeneCounts )
g_index=/home/changyj307/changyj307/genome_index_p14_v45 # STAR genome index
out_dir=/home/changyj307/changyj307/77_RNAseq/align_rsem
files=/home/changyj307/changyj307/77_RNAseq/hs23839/clean_all_1
for i in $(ls $files/*_R1.fastq.gz | sort -u)
do
STAR \
--genomeDir $g_index \
--readFilesIn ${i} ${i/_R1/_R2} \
--runThreadN 20 \
--twopassMode Basic \
--outSAMtype BAM SortedByCoordinate \
--outSAMunmapped Within \
--readFilesCommand zcat \
--genomeFastaFiles $g_index/GRCh38.p14.genome.fa \
--quantMode TranscriptomeSAM \
--outFileNamePrefix ${out_dir}/${i/$files/} ; done;
The Log.out showed:
STAR --genomeDir /home/changyj307/changyj307/genome_index_p14_v45 --readFilesIn /home/changyj307/changyj307/77_RNAseq/hs23839/clean_all_1/ip-D-M-L-18D-1_22C5KWLT3_L5_clean_R1.fastq.gz /home/changyj307/changyj307/77_RNAseq/hs23839/clean_all_1/ip-D-M-L-18D-1_22C5KWLT3_L5_clean_R2.fastq.gz --runThreadN 20 --twopassMode Basic --outSAMtype BAM SortedByCoordinate --outSAMunmapped Within --readFilesCommand zcat --genomeFastaFiles /home/changyj307/changyj307/genome_index_p14_v45/GRCh38.p14.genome.fa --quantMode TranscriptomeSAM --outFileNamePrefix /home/changyj307/changyj307/77_RNAseq/align_rsem//ip-D-M-L-18D-1_22C5KWLT3_L5_clean_R1.fastq.gz
STAR version: 2.7.10a compiled: 2022-01-14T18:50:00-05:00 :/home/dobin/data/STAR/STARcode/STAR.master/source
Mar 11 01:02:40 ..... started STAR run
Mar 11 01:02:40 ..... loading genome
STAR --genomeDir /home/changyj307/changyj307/genome_index_p14_v45 --readFilesIn /home/changyj307/changyj307/77_RNAseq/hs23839/clean_all_1/ip-D-M-L-18D-1NP-1_22C5KWLT3_L6_clean_R1.fastq.gz /home/changyj307/changyj307/77_RNAseq/hs23839/clean_all_1/ip-D-M-L-18D-1NP-1_22C5KWLT3_L6_clean_R2.fastq.gz --runThreadN 20 --twopassMode Basic --outSAMtype BAM SortedByCoordinate --outSAMunmapped Within --readFilesCommand zcat --genomeFastaFiles /home/changyj307/changyj307/genome_index_p14_v45/GRCh38.p14.genome.fa --quantMode TranscriptomeSAM --outFileNamePrefix /home/changyj307/changyj307/77_RNAseq/align_rsem//ip-D-M-L-18D-1NP-1_22C5KWLT3_L6_clean_R1.fastq.gz
STAR version: 2.7.10a compiled: 2022-01-14T18:50:00-05:00 :/home/dobin/data/STAR/STARcode/STAR.master/source
Mar 11 01:03:27 ..... started STAR run
Mar 11 01:03:28 ..... loading genome
The err.out showed:
EXITING because of FATAL ERROR: cannot insert sequence on the fly because of strand GstrandBit problem
SOLUTION: please contact STAR author at https://groups.google.com/forum/#!forum/rna-star
Many thanks for your helping!
YuJung
Hi YuJung,
Please remove --genomeFastaFiles
from the mapping command, you do not need it since you already used it in the genome generation step.