I was using STAR-RSEM to quantify isoforms (primary input fastq files). Recently I have received Aligned.sortedByCoord.out.bam from the collaborators. However, I can't use these files as an input in RSEM. I have used samtools fastq to convert bam to fastq files (paired). I have used the preexisting data set for the preliminary analysis.
samtools: samtools fastq -1 Aligned.sortedByCoord.out.R1.fastq -2 Aligned.sortedByCoord.out.R2.fastq -O -t Aligned.sortedByCoord.out.bam
STAR alignment using fastq R1&R2 (samtools generated fastq files from Aligned.sortedByCoord.out.bam) generated higher unmapped reads %
Number of input reads | 23575225 Average input read length | 186 Uniquely mapped reads number | 5556353 Uniquely mapped reads % | 23.57% % of reads unmapped: too short | 69.93%
STAR alignment using original fastq R1&R2 generated higher Uniquely mapped reads %
Number of input reads | 23575225 Average input read length | 186 Uniquely mapped reads number | 20455386 Uniquely mapped reads % | 86.77% % of reads unmapped: too short | 7.45%
I do not know why there is a huge difference. I do not know if I am missing any steps?