Project overview
The safety of blood and blood-based products is a critical priority in blood transfusion and organ transplantation. The Duncan Laboratory at the Center for Biologics Evaluation and Research, FDA, has developed a proof-of-concept, multiplexed detection, and identification platform- RPM-BBPv.2, that interrogates clinical samples for the presence of viral, bacterial, and protozoan pathogens [1].
The bbp_i2o data processing pipeline
The bbp_i2o was originally implemented to process data from the RPM-BBPv.2 platform and provide a readout of the list of pathogens likely present in a test sample. The program is adapted to take the sequence file (a text file of the base calls for all the tiles on the microarray) from a run of RPM-BBPv.2 as its only user-defined input. It outputs the following files: (a) List of tile sequences with C3 scores greater than or equal to 20.0 that were aligned against the nt database (file name: .blast.in.fst. Can be read with Notepad) (b) The BLAST report listing the top hits for each tile, along with control results (file name: .blast.report. Opens with Notepad) (c) The detailed BLAST report in HTML format (file name: .blast.html) (d) A calculation of the rank order of the species hits in the BLAST results (file name: .ID.report. Can be opened with Notepad) (e) A file containing species rejected for unknown names (file name: .Unrecognized.Names. Can be read with Notepad)
Installation/Dependencies
The bbp_i2o program is written in Perl. To run the program, you will need to install the following i. Perl modules: (a) File::Path (b) List::MoreUtils ii. Packages: the BLAST, sequence alignment package from NCBI iii. Databases: nt, the non-redundant NCBI nucleotide collection iv. Additional files: tile_contaminating_sequences: a mask file with coordinates on each sequence tile to be masked; ScientificNamesList - a file that contains the list of less specific scientific names. These files are included with this version of bbp_i2o.
After downloading bbp_i2o onto your machine, open the program with a text editor and update: i. $BLASTN to the full path of the blastn module of your BLAST package. ii. $BLAST to the full path to the nt database on your machine. iii. $TILECONTFILE to the full path to the tile_contaminating_sequences file on your machine. iv. $ScientificNamesFile to the path of ScientificNamesList file on your machine. The bbp_i2o program has examples of paths we use in our laboratory.
Running bbp_i2o
The program is run as perl bbp_i2o . It takes 5 -10 minutes to complete running.
- Kourout, M., et al., Multiplex detection and identification of viral, bacterial, and protozoan pathogens in human blood and plasma using a high-density resequencing pathogen microarray platform. Transfusion, 2016. 56(6 Pt 2): p. 1537-47.