Error messages when normalizing with DESeq2 and CSS

[kathfi]

Dear all,

I wonder if anyone could help me with the following: I would like to normalize an out.biom table using CSS, and I keep getting the following error message:

Traceback (most recent call last): File "/usr/local/bin/normalize_table.py", line 157, in main() File "/usr/local/bin/normalize_table.py", line 140, in main normalize_CSS(input_path, out_path, output_CSS_statistics) File "/usr/local/lib/python2.7/dist-packages/qiime/normalize_table.py", line 38, in normalize_CSS run_CSS(temp_fh.name, out_path, output_CSS_statistics=output_CSS_statistics) File "/usr/local/lib/python2.7/dist-packages/qiime/normalize_table.py", line 76, in run_CSS app_result = rsl(command_args=command_args, script_name='CSS.r') File "/usr/local/lib/python2.7/dist-packages/qiime/util.py", line 1968, in _call (exit_status, command, stdout, stderr)) burrito.util.ApplicationError: Unacceptable application exit status: 1 command: cd "/home/qiime/Desktop/sandlance/alldata/Normalization/"; R --slave --args --source_dir /usr/local/lib/python2.7/dist-packages/qiime/support_files/R -i /tmp/QIIME-normalize-table-temp-table-5uGkcm.biom -o otu_table_silvamod_css.biom -s otu_table_silvamod_css_CSS_statistics.txt < /usr/local/lib/python2.7/dist-packages/qiime/support_files/R/CSS.r stdout:

stderr: Loading required namespace: biomformat Found more than one class "file" in cache; using the first, from namespace 'BiocGenerics' Found more than one class "file" in cache; using the first, from namespace 'BiocGenerics' Error in cumNormStatFast(obj) : Warning sample with one or zero features Calls: CSS -> cumNormStatFast Execution halted_

I am using an OTU-table that has taxonomy already assigned and samples with <1000 reads excluded, hence I should not have a sample with 1 or 0 features as the message indicates. Am I overlooking something else? Why does this message persist?

Another issue I am experiencing is with DESeq2, maybe you can help me here, too? I am executing these 3 commands to normalize, create a .tab file with taxonomy info and then reassign the taxonomy to the normalized file:

biom convert -i otu_table_silvamod.biom -o otu_table_silvamod.tab --to-tsv --header-key taxonomy normalize_table.py -i otu_table_silvamod.biom -o otu_table_silvamod_deseq.biom -a DESeq2 -z biom add-metadata -i otu_table_silvamod_deseq.biom -o otu_table_silvamod_deseq_tax.biom --observation-metadata-fp otu_table_silvamod.tab --observation-header OTUID,taxonomy

These commands work, however taxonomy = 0 in all but 1 case in the file “otu_table_silvamod_deseq_tax.biom”. Why may this be the case? I have created PCoA plots, PCA plots, rarefaction curves etc. with the non-rarefied samples and they look fine.

Any help on these two issues would be greatly appreciated.

Many thanks in advance, Katharina

[gregcaporaso]

Can you please post your question to the QIIME Forum? We try to address these types of technical support questions there, as it's then easier for others to find answers when they have the same questions. Thank you!

[kathfi]

Dear Greg,

unfortunately I don't have permission to post in the forum that you indicate, hence maybe it is possible to address this issue here, or let me know how I can gain access?

many thanks Katharina

[gregcaporaso]

You'll just need to create a Google Groups account or login with an existing Google account.