Hi, I used tophat2 to align paired-end reads to Arabidopsis genome and I want to use the unmapped.bam file to obtain the unmapped reads so I can align to a transgene. The output gives the name of the reads without the paired-end information, so I tried several scripts to extract the reads from the fastq files present on the unmapped.bam, but they did not work. I tried using HISAT2 and bowtie2, but the statistics are completely different, such as no concordant alignment when tophat2 would give me above 95% alignment rate and above 90% of concordant alignment. I want to go back to tophat2, but I need the unmapped reads.
For me the best would be continue using tophat but having a way to filter the fastq files with the unmapped.bam. Does anyone have an idea of how to do that?
Here is the code I used for the alignments: tophat2
tophat -o Control/Control_5 --library-type fr-firststrand -r 60 --mate-std-dev 50 index/TAIR10 CNT-5P_R1.fastq CNT-5P_R2.fastq
hisat2 --no-mixed -X 310 -x sat_index/hisat_tair -1 CNT-5P_R1.fastq -2 CNT-5P_R2.fastq -S Control/Control_5.sam --summary-file Control/Control_5.txt
bowtie --no-mixed -I 210 -X 310 -x index/TAIR10 -1 CNT-5P_R1.fastq -2 CNT-5P_R2.fastq -S Control/Control_5.sam