Hello everybody,
I am really wondering about the results I get at the end of my analysis. So I hope to get some feedback from the community :)
I followed the tutorial located at
http://qiime.org/tutorials/tutorial.html to create a plot with stacked bar charts for gut microbiome analysis. This way I compare the contained 6 barcoded samples in my fastq file with each other. The commands I used were:
#step 1
validate_mapping_file.py -m map1.txt -o validate_map
#step 2
convert_fastaqual_fastq.py -c fastq_to_fastaqual -f file.fastq -o fastaqual
#step 3
split_libraries.py -m map1.txt -f fastaqual/file.fna -q fastaqual/file.qual -o split_library_out/ -b 13 -l 140 -z truncate_only
#step 4
pick_de_novo_otus.py -i split_library_out/seqs.fna -o otus/
#step 5
assign_taxonomy.py -i otus/rep_set/seqs_rep_set.fasta -m rdp -o rdp_assigned_taxonomy
#step 6
make_otu_table.py -i otus/uclust_picked_otus/seqs_otus_txt -t rdp_assigned_taxonomy/seqs_rep_set_tax_assignments.txt -o L7_otu_table.biom
#step 7
summarize_taxa.py -i L7_otu_table.biom -o L7_taxonomy_summary/ -L 7
#step 8
plot_taxa_summary.py -i L7_taxonomy_summary/L7_otu_table_L7.txt -o L7_taxonomy_plot/
I used the same procedure to process a second fastq file with 6 other samples. So far, the pipeline works well.
My problem is now that I would like to compare 2 samples from different biom files in a stacked bar chart with each other. I googled around a bit and these commands looked good to me:
# Split biom file belonging to file.fastq (created at step 6 above) by SampleID
split_otu_table.py -i L7_otu_table.biom -m map1.txt -f SampleID -o split_by_sample
# The same for file2.fastq
split_otu_table.py -i L7_otu_table.biom -m map2.txt -f SampleID -o split_by_sample
# Merge 2 biom files containing the samples I want to compare
merge_otu_tables.py -i otu_table.file.fastq.biom,otu_table.file2.fastq.biom -o merged_otu_table.biom
# Now I follow the above procedure starting at step 7 to create a bar chart with new biom file
summarize_taxa.py -i merged_otu_table.biom -o new_taxonomy_summary/ -L 7
plot_taxa_summary.py -i new_taxonomy_summary/merged_otu_table_L7.txt -o new_taxonomy_plot/
I end up with a new plot, containing 2 bar charts; but I am wondering that the percentages in one of the two charts are completely different compared to its initial version. To my mind I should get a plot with 2 bar charts, containing identical percentages like their initial version.
Am I doing something wrong or do I miss a step here? I really can't imagine why values change in my new plot.
Any advise is really appreciated :)
Patrick