Actually I map raw reads to CDS of Draft genome where all genes CDS is not present.
I used below cammand lines for pair end read mapping to CDS and want to extract unmapped pair end reads in which BOTH READS of Pair not able to map to CDS.
But when I am running last cammnd it show some warning on screen: but it write R1 and R2 READS in two separete files. is it ok?
WARNING: Query PC168224:127:C98A1ANXX:1:2316:21235:23135 is marked as paired, but its mate does not occur next to it in your BAM file. Skipping. WARNING: Query PC168224:127:C98A1ANXX:1:2316:21236:13229 is marked as paired, but its mate does not occur next to it in your BAM file. Skipping. *WARNING: Query PC168224:127:C98A1ANXX:1:2316:21237:66490 is marked as paired, but its mate does not occur next to it in your BAM file. Skipping.
For BWA indexing
/home/yog/software/bwa-0.7.15/bwa index -p Radish_index Radish_cds.fasta
/home/yog/software/bwa-0.7.15/bwa mem Radish_index forward_216_5W_Ca1.fastq reverse_216_5W_Ca1.fastq >216_5W_Ca1.sam
Convert Sam to BAM
/home/yog/software/samtools-1.3.1/samtools view -bS 216_5W_Ca1.sam > 216_5W_Ca1.bam
Extract Unmapped Reads:
/home/yog/software/samtools-1.3.1/samtools view -b -f 4 216_5W_Ca1.bam > unmap_216_5W_Ca1.bam
To convert bam to Fastq File:
/home/yog/software/samtools-1.3.1/samtools sort -n unmap_Ca1.bam -o unmap_216_5W_Ca1._resorted /home/yog/software/bedtools2/bin/bamToFastq -i unmap_Ca1._resorted -fq Unmap_Ca1_read1.fastq -fq2 Unmap_Ca1_read2.fastq