Automated DNA barcode assembly, quality control, and annotation
DNABarcodeReader is designed to automate the steps between obtaining raw DNA sequence data and running phylogenetic/population genetic analyses. The steps include: (1) Creating individual assemblies for each sample in a dataset; (2) Generating quality control reports; (3) Querying local and WWW BLAST databases annotate the barcode with taxonomic information; and (4) Outputting DNA barcode sequence data into a text file in different formats for downstream analyses (e.g., FASTA).
#Prerequisites
##Python
DNABarcodeReader is is writen in Python verstion 2.7+. So, you will need to download and install Python if you don't have it already. Information on Python, including tutorials can be found here. Installers for Linux, Mac, Windows can be downloaded here. DNABarcodeReader has NOT been tested in Windows. Feedback of its use in Linux and Windows would be much appreciated. DNABarcodeReader was developed using version 2.7.5 of Python.
##BioPython
Most of the features of DNABarcodeReader are written using the standard modules supplied with a standard Python installation. Two additional modules will be needed, however. One is BioPython, which includes some of the basic code used to generated sequence output files (e.g., in FASTA format), and some of the functionality needed to run BLAST queries. All the information you will need on BioPython can be found here. Download and installation instructions can be found here. DNABarcodeReader was developed using version 1.62 of BioPython.
##ZODB - native object database for Python
The second extra module is ZODB, which provides an easy interface to create a database of Python objectcs. The database ensures persistence of work, and allows one to grow a project by simply adding new raw data, rather than having to restart from scratch. All information required to download, install, and run ZODB can be found here. DNABarcodeReader was developed using version 3.10.3 of ZODB.
##Phred, Phrap, and Consed
A core process in generating DNA barcodes consists of producing assemblies of raw DNA sequence data for each individual sample. One of the best assemblers out there, in particular for Sanger sequence data - and soon for NGS type data, is formed by the Phred, Phrap, Consed trio. Phred (by Brent Ewing) is a base-caller that takes as input raw chromatograms and outputs empirically validated quality scores; Phrap (by Phil Green) is an assembler, which takes the ouptut from Phred as an input; and Consed (by David Gordon) is an assembly visualisation and editting tool. The three tools work on all major computing platforms. All three tools are the intellectual property of the University of Washington. Academic licenses are available for free, but commercial licenses incur a cost. Instructions on how to obtain a license, download, install, and use Phred, Phrap, and Consed can be found here.
Only Phred and Phrap are essential.
Consed is only needed as a post-processing tool, to examine DNA barcodes that do not pass QC. Even though Consed is not needed, we highly recommend its use.
###References Ewing, B., Hillier, L., Wendl, M., and Green, P., 1998, "Basecalling of automated sequencer traces using phred. I. Accuracy assesment", Genome Research 8: 175-185.
Ewing, B. and Green, P., 1998, "Basecalling of automated sequencer traces using phred. II. Error probabilities", Genome Research 8: 186-194.
Green, P., 1994, Phrap, unpublished. http://www.phrap.org
Gordon, D., Abajian, C., and Green, P., 1998, "Consed: A grapical tool for sequence finishing", Genome Research 8:195-202.
##PolyPhred
DNA barcodes are generally designed around a mitochondrial or chloroplast gene. These are haploid genomes, and thus carry the special assumption that there should be no variation within individuals at a particular site. This is convenient from the barcode perspective. Each sample should have a string of unambiguous bases that represent its DNA barcode. When variation does occur, two possible explanations are possible: (1) there is experiment error (e.g., sample was contaminated with DNA from another sample); or (2) there is something biologically interesting happening (e.g., blood sample is infected with multiple lineages of a parasite). DNABarcodeReader won't help identify which of these two has occurred, but it will allow you to optionally use PolyPhred (by Debrah Nickerson) to assess the chance that a sample contains multiple barcodes. PolyPhred examines chromatograms for signatures of overlapping peaks.
As in Phred,Phrap, and Consed, we do not distribute PolyPhred with DNABarcodeReader. PolyPhred is the intellectual property of the University of Washington. Academic licenses are available for free, but commercial licenses incur a cost. Instructions on how to obtain a license, download, install, and use here.
###References Nickerson, D.A., Tobe, V.O., and Taylor, S.L, 1997, "PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing", Nucleic Acids Research, 25: 2745-2751.
##NCBI BLAST
In some cases, the user may have a specific database that they wish to use to annotate their barcodes. For instance, the user might have a database of barcodes of known organisms in his or her study area; or, they may have a database of organisms that are listed under CITES Appendix I. To be able to query these databases, the user will require at least a FASTA file with all the barcode sequences of interest, with suitable annotation, and a local copy of BLAST software. Instructions on how to obtain a copy and install BLAST locally can be found here. All the command line instructions for running BLAST locally can be found here. However, the user does not need to know any of these commands if he/she wants to use DNABarcodeReader as is. They simply need to make sure it is installed.