Hi guys, I am relatively new to alignment tools and bioinformatics in general. I have seen several questions on the forums pertaining to my question, but after following through on the answers, I can't seem to get the right result. Basically, I am trying to find where a plasmid tDNA insertion of known sequence is in an arabidopsis thaliana genome. My plan was to use split alignment methods for this. I have a reference genome consisting of 5 chromosomes in fasta format, to which I apended the known plasmid contruct sequence with the header ">Plasmid". I have paired end reads that I want to align to multiple places in the "Genome+Plasmid" reference with the hope that I can later use BLAST to find which reads align to one of the chromosomes as well as the plasmid. With this information I hope to use BLAST to determine the gene that the tDNA insertion is next to. It is my understanding thus far that I want split or "chimeric" reads, but not supplementary reads. I have a good understanding of how to use BLAST, but I am having difficulty finding split alignment reads. So far, I have tried bwa-mem, segemehl, and bowtie2 on ubuntu, but none have worked. I'm not sure if this is because I am incorrectly setting parameters or some other issue. Also, once the split alignments are found and marked, I'm not sure how to extract them. I also am unable to open .sam files, (not sure if people normally can?) If anyone could show me a step-by-step process with one of the above alignment tools (or a new tool if necessary) of how to find and extract split reads, this help would be greatly appreciated. If I am unclear in any way, please let me know. Thanks
segemehl commands- resulted in no reads ./segemehl.x -i refindex.idx -d ref.fas -q reads1.fasta -p reads2.fasta > output.sam samtools view -bS -o output.bam output.sam samtools sort output.bam sorted.bam samtools view -h -o sorted.sam sorted.bam ./testrealign.x -d ref.fas -q sorted.sam -n
bowtie2- not sure how to extract bowtie2 -x ref.fas.bowtie2 -1 reads1.fq.gz -2 reads2.fq.gz -S output.sam --local
bwa mem bwa mem ref.fas reads1.fq reads2.fq > output.sam samtools view output.sam I tried several different commands after this but I think I was going in the wrong direction. I believe I tried the virst command with the -M function as well. how can I extract split reads from this point?