How to get the extended unaligned bases at the end of the read in reference based genome assembly?

Question

[Kumar]

Hi all, I had done a illumina read mapping based on a particular gene sequence (reference sequence), which is highly repetitive in nature by using the following command

bowtie2-build -f ref_gene.fasta ref_index && bowtie2 -p 4 -N 1 -t -x ref_index -1 genome_R1.fastq -2 genome_R2.fastq -S tal.sam && samtools view -bS tal.sam | samtools sort - -o tal.bam && samtools mpileup -uf ref_gene.fasta tal.bam | bcftools call -c | vcfutils.pl vcf2fq > cns.fastq && seqtk seq -aQ64 -q20 -n N cns.fastq > contig.fasta

It is works perfectly fine and yielded exactly same length of mapped sequence as ref_gene. However, I need to get both end (reverse and forward end) extended overlapping reads in the assembled contig (Please see the image for better understanding) Is there any way to do it the same? Thanks in advance.

Answer 1

[WouterDeCoster]

If I understood your question correctly you are looking at unaligned bases at the end of reads. That means you have to extract the soft clipped bases. Googling for that brings up this thread: extracting the soft clipped seq only from a sam file

[Kumar]

Thank you @WouterDecoster for your suggestion and giving me the proper technical word for the same.

[Kumar]

Dear @WouterDeCoster, the tool you have suggested only gives the mapped/aligned reads (exactly same length as reference). It is not giving the unaligned overlapped reads.

[Devon Ryan]

I suspect you're looking at the wrong file.