I ran an Illumina 2 x 250 PER on PCR amplicons and received the data demultiplexed by the Index on Read 2. The read 2 sequences were not quite long enough to reach the UMIs (barcodes) at the end of each sequence, so I then ran PEAR to assemble read 2 and read 1. The output clearly shows the additional nucleotides added to the end of read 2 with the barcodes present. However, when I use the Barcode Splitter tool on Galaxy, it does not even show the assembled file as a choice for “Library to Split.” Why is this not an option, ie., what have I done wrong? Is there an alternative way to achieve the result I am desiring?
I know that I can simply barcode split the reverse complement of the read 1 files, but it seems like I will be wasting the sequence information provided by read 2. Also, in a future experiment, I am going to have non-indexed sequence data. In that case, I will need the barcode information provided by read 1 plus the sequence information provided by read 2 (the amplicon is greater than the 2 x 150 PER we will be doing).
Thank you for your help. Michael