Analyzing 454 Sequencing

• Qiime Website
• Qiime Tutorial

General Steps

1. Check Mapping File
2. Split Library
3. Pick OTUs
4. Pick Representative Sequence
5. Align (Optional to do now or after building the table)
6. Assign Taxonomy
7. Build OTU Table
8. Align (if not performed before assigning the taxonomy table)

Notes

• When running natively on MacQiime: must use macqiime (with a space after) before typing in code in the terminal window
  • macqiime check_id_map.py -m Fasting_Map.txt -o mapping_output -v
• When running natively: $q/
  • $q/check_id_map.py -m Fasting_Map.txt -o mapping_output -v

Combine Data Sets

If you have two .fna and .qual files that you want to combine and you have the same barcodes for both data sets do the following:

1. Combine .qual files (simple copy and paste into a new file)
2. Combine .fna files
   1. First you must edit each .fna file

• • Copy and paste the file into Excel
  • It should show up with a title <G234 etc. followed by the sequences in the following cells
  • Ex. A1 = title A2 = sequence A3 = continuing sequence
  • In the next column type =IF(ISNUMBER(SEARCH(“*>*”,A1)),”G”,””)
  • This will put a G in the column B if column A has > (title)
  • Shift column B down 1 cell
  • In column C, =CONCATENATE(B1,A1)
  • This will add a letter at the beginning of each sequence
  • Save

Repeat with the second .fna file but be sure to use a different letter than G

Apply the same correction to the barcodes in your mapping files.

Creating Ordination Plot

1. Align Sequences
2. Filter
3. Make Phylogenetic Tree
4. Rarify

Analysis of beetle bacteria

• checking the mapping file:: check_id_map.py -m Beetle_map_NEW.txt -o mapping_test
• split_libraries.py -m Beetle_map_NEW.txt -f 5.TCA.454Reads.fna -q 5.TCA.454Reads.qual -o split_library_output
• pick_otus.py -i seqs.fna -o picked_clustered_otus/ #kept at default: similarity 0.97; reverse strand matching did not change the output
• picking representative sequences:: pick_rep_set.py -i picked_clustered_otus/seqs_otus.txt -f seqs.fna -o rep_set.fna #all default (the most common one)
• alignment by MUSCLE:: align_seqs.py -i rep_set.fna -m muscle -o alignment/
• taxonomy assignment:: assign_taxonomy.py -i rep_set.fna -m rdp
• make_otu_table.py -i picked_clustered_otus/seqs_otus.txt -t rdp22_assigned_taxonomy/rep_set_tax_assignments.txt -o OTU_table_NEW.txt # making OTU table
• rarefaction to three different levels: 41 (to get a few crassiusculus), 122 (to retain more mesonotal), 330.
  • example with 41: single_rarefaction.py -i OTU_table_NEW.txt -o rarefaction/rarefaction_41.txt -d 41
• making preferences file:: make_prefs_file.py -m Beetle_map_NEW.txt -b”species,locality,species&&locality” -k white -o prefs_file.txt
• make phylogeny using Fasttree:: make_phylogeny.py -i alignment/rep_set_aligned.fna -o rep_phylogeny.tre
• beta diversity matrix of similarity
  • with full matrix: beta_diversity.py -i OTU_table_NEW.txt -t rep_phylogeny.tre -o b_diversity/
  • with rarefied matrices, e.g.: beta_diversity.py -i rarefaction/rarefaction_41.txt -t rep_phylogeny.tre -o b_diversity/
• PCoA batch: principal_coordinates.py -i b_diversity/ -o PCoA/
• making PCoA plots: make_2d_plots.py -i PCoA/pcoa_unweighted_unifrac_full_table.txt -m Beetle_map_NEW.txt -p prefs_file.txt -o 2d_plots/full_unweighted/
• making UPGMA cluster: upgma_cluster.py -i b_divers_condensed/unweighted_unifrac_rarefaction_41.txt -o b_divers_condensed/UPGMA_output.txt
  • getting support for UPGMA nodes:
  • multiple rarefactions: multiple_rarefactions.py -i b_divers_condensed_batch/OTU_table_NEW_condensed.txt -o b_divers_condensed_batch/ -m 41 -x 330 -s 10 -n 2
  • beta_diversity.py -i b_divers_condensed_batch/ -t rep_phylogeny.tre -m unweighted_unifrac -o b_divers_condensed_batch_b_divers/
  • upgma_cluster.py -i b_divers_condensed_batch_b_divers/ -o b_divers_condensed_UPGMAs/
  • tree_compare.py -m b_diversity_condensed/master/UPGMA_master.tre -s b_divers_condensed_UPGMAs/ -o b_diversity_condensed/

WITH ZEROS for beetle without bacteria – didn’t work, the ones without bacteria drive all patterns

• bray_curtis and Binary jaccard:: beta_diversity.py -i OTU_table_incl_zeros.txt -m bray_curtis,binary_jaccard -o b_diversity_incl_zeros/