BIR pipeline steps and subsequent output files description STEP 1: BLAST search

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1 Lifeportal (Brief description) The Lifeportal at University of Oslo ( is a Galaxy based life sciences portal lifeportal.uio.no under the UiO tools section for phylogenomic analysis, population genetics and high-throughput sequence analysis. The main advantage of Lifeportal lies in an access to a parallel computational resource that enables demanding computations. Therefore, this resource is designed for large, time consuming computations rather than for an interactive use. The Lifeportal use is free but requires a user account registration using the link Register for BIR access from lifeportal homepage. BIR Overview BIR - A pipeline for several initial tasks involved in phylogenetic reconstructions. In particular, it is useful for multi-gene phylogenies. It screens non-annotated sequences aganist seed alignments and selected eukaryotic genomes (protein sequences) using BLAST. It constructs single gene alignments (MAFFT), trims alignments (Gblocks or trimal), performs phylogenetic analysis of each seed alignments (FastTree or RAxML) and predicts orthologs (coco-cl). Input files BIR requires a query sequence file and seed alignment or sequence file, both compressed using ZIP separately and in FASTA format (Figure 1). Each ZIP file may contain one or several files. Figure 1: Snapshot of input file selection panel BIR pipeline steps and subsequent output files description STEP 1: BLAST search The first step starts with BLAST searches: query files are matched against all the seed alignment files provided. BLAST parameter and BLAST output parsing criteria are specified on the webinterface (Figure 2) Sequence type (nucleotide or amino-acid) has to be specified. 1

2 All sequence files provided in alignment files must be either nucleotide or amino acid. Query and alignment files must be nucleotide for BLASTN. Query and alignment files must be amino acid for BLASTP. Query files must be nucleotide and alignment files must be amino acid for the third option of (transeq and BLASTP). Figure 2: Snapshot of BLAST search parameters. Based on the quality criteria, the sequences from query files are added to seed alignments. If the sequences are matched against more than one seed alignment then it is added to all the seed alignment files. 2

3 Output file description (Files created in STEP 1) All result files described below are written in a directory named Additional_files. 1. ParamInfo.txt: This file lists all the command line parameters and steps included and executed for analysis. Brief statistics of number of files and number of sequences in each files uploaded as query and seed alignments. It also list total number of sequences added (from query and genome) to any alignment files. Invalid parameter selection or value entered or error encountered leading to unsuccessful of pipeline are also listed here. 2. RawBlastOutput.txt: Raw BLAST output for query sequences in text format. 3. RawBlastGenomeOutput.txt: Raw BLAST output for genome sequences in text format. 4. Stats_table_Q.txt: This tab delimited text file list summary of sequences from query file added to seed alignment files. The order of the column is 1)query sequence name, 2)query file name, 3)number of seed alignment files where is query sequences are added, 4)number of sequences above cut off set by the user / total number of sequeneces in seed alignment file, and seed alignment file name. 5. Stats_table_G.txt: This tab delimited text file list summary of sequences from selected genome added to seed alignment files. The order of the column is 1)seed alignment file name, 2)genome, 3)identity percentage of added sequences from genomes, 4)GI. 6. BlastPars_Stats_Q.txt: This file lists all sequences from query files passing the quality criteria (query coverage, sequence identity percentage, bit score and e-value criteria) along with their BLAST search result statistics (query length, alignment length, identity percentage, overlap percentage, bit score, e-value). 7. BlastPars_Stats_G.txt: This file lists all sequences from genome files passing the quality criteria (query coverage, sequence identity percentage, bit score and e-value criteria) along with their BLAST search result statistics (query length, alignment length, identity percentage, overlap percentage, bit score, e-value). 8. QuerySeqAdded.fasta: Sequences from query files that are added to any seed alignment files are written in FASTA format for subsequent external analysis. If any sequences from query files are added in more than one seed alignment, it is listed only once. 9. GenomeSeqAdded.fasta: Sequences from genome files that are added to any seed alignment files are written in FASTA format for subsequent external analysis. 3

4 STEP 2: Re- align alignment files BIR uses MAFFT for aligning sequences (Figure 3). The newly added sequences in each seed alignment can either be realigned or aligned to exiting alignment (preserving the original alignment as provided by the user). Note: Must activate Re-align step for further step. Figure 3: Snapshot of alignment parameters. Output file description (Files created in STEP 2) The output files created in STEP 2 are stored in directory named Aligned. 1. SeedAlignment.fasta: Aligned seed alignment files. Sequences added from query file and genomes are included. STEP 3: Removal of ambiguously aligned characters trimal and Gblocks implemented in BIR may be used to remove poorly aligned positions or regions of an alignment (Figure 4 and Figure 5). The removal can be stringent (default) or relaxed (select the checkbox to override default). 4

5 Figure 4: Snapshot of Gblocks parameters. Figure 5: Snapshot of trimal parameters. Output file description (Files created in STEP 3) Gblocks output files are stored in a directory named Gblocks. 1. SeedAlignment.fasta-gb or SeedAlignment.fasta-Al: After removing poorly aligned positions of an alignment file (Gblocks/trimAl output files in FASTA format). 2. SeedAlignment.fasta-gb.htm or SeedAlignment.fasta-Al.html: A HTML file with figure of selected positions underlined with a heavy blue line and highlighted conserved positions. The parameters and color coding details can be accessed at following web links for 5

6 Gblocks - trimal - STEP 4: Phylogenetic inference FastTree/RAxML an approximately-maximum-likelihood method may be used for inferring trees for single gene alignments (Figure 6). Figure 6: Snapshot of FastTree options. Output file description (Files created in STEP 4) FastTree output files are stored in a directory named FastTree. RAxML output files are stored in a directory named RAxML. 1. SeedAlignment.fasta-gb-marked: The -marked extension files includes information about the sequences added from query files and genome files along with paralogs annotation. The fasta headers are extended by _*Q (sequences added from query files ), _*G (sequences added from genome files), and _*C (sequences predicted to be paralogs using COCO-CL). 2. SeedAlignment.mtree: Tree file for each seed alignment file in Newick format. Sequences from query and genome file added to seed alignment file are marked with _*Q and _*G respectively. The coco-cl predicted paralogs are marked _*C. There are many phylogenetic tree viewer tools that accepts Newick format to view tree files generated. Links for tree viewing tools are : FigTree ( 6

7 TreeView ( MEGA ( FastTree - RAxML - Step 5: Ortholog prediction (COCO-CL) coco-cl output files are stored in a directory named coco-cl. COCO-Paralogs_table.txt: This tab delimited text file list summary of paralogues in each seed alignment files. The order of the column is 1) seed alignment file name, 2) number of paralogs found, 3) fasta header of potential paralogs predicted using coco-cl SeedAlignment_fas-gb.cls: Raw output file of coco-cl. Cluster 1 list all the orthologs sequence header while Cluster 2 list all paralogs. coco-cl - 7