I extracted the specific region from a BAM file generated by aligning fastq (Illumina/whole genome sequencing) on hg38 and converted to corresponding fastq reads of that region by Picard (SamTofastq) and try to realign to another (my own) reference sequence by bwa mem. But, it seems that bwa mem cannot understand the sequencing is as paired end since it frequently says “skip orientation FF as there are not enough pairs” during the mapping, it also happened for other orientations (FR, RF, and RR); however, there is 1259352 paired read. Subsequently, the properly paired percentage is very low. Could you please kindly help me out how I can solve the problem?
Edit and update: I also found that during the conversion of bam to fastq by SamTofastq (picard), it returned me: SAM validation error: ERROR: Found 13548 unpaired mates. I think, it may not be a real problem; actually, it seems that Picard just consider paired read to creat Fastq file. So, the real problem is with bwa mem, yes? Please kindly share your idea with me?
Here is the used commands:
samtools sort file.bam sammtools index file.bam file.bam.bai Extracted the region of interest: samtools view -h -b file.bam chr6:28,510,120-33,480,577 > extracted_new.bam Create fastq java -jar SamToFastq.jar I=extracted_new.bam F=file_1.fastq F2=file_2.fastq Alignment by BWA-MEM bwa index my_reference.fasta bwa mem -L 10000 -a my_reference.fasta file_1.fastq F2=file_2.fastq > output.sam