This is my first time performing NGS alignment. I am using command line tools. What I did so far, was to first use cat to combine all chromosomes of my reference genome. I then indexed the genome with bwa (-a bwtsw). I then de-multiplexed and sorted into separate FASTQ files by barcode using FASTX Toolkit's Barcode Splitter. Next, I used FASTX trimmer to remove 75bp of barcode, primer, and transposon sequence (I am over trimming, but I started with 300 bases). I then aligned with bwa, trying both the mem and samse algorithms. Then, with samtools, I converted to BAM format with the view command, sorted, and indexed. In viewer programs such as Tablet, IGV, and BamView, I either saw no reads/blank or, in the case of Tablet, I saw that it had categorized reads by chromosome, but clicking on a chromosome showed nothing. This is true for the output of both algorithms. Not sure if it is worth noting, but in Tablet, the mismatches column for each chromosome all show a "?". What could go wrong along the way?
Thanks for your help.