16s some qiime questions

[rajal.debnath]

I have 0ne 16s sample (without barcodes, as this is only one sample) sequenced by 454. I want to use qiime to analyze it:
1. Which step do I need to start from ? It is only one sample so do I need the steps of preparing the mapping file and the "assign sample to multiplex reads"?
If I do need them, how to do this in case this is only one unbarcoded sample? If I do not need it, and begin from the "pick OTU" step, I will lose the "filtering step" which is part of the " assign samples to multiplex reads", won't I?
2.Can I skip the "align OTU sequences" and the "filter alignment" and "phylogenetic tree" if all I am interested in is the OTUS whixh were found in the sample and the abunduncies in this only sample?
3.I made an OTU table, but when I tried to view the statistics of it via the command :" print_biom_table_smmary.py" I received a message that the command does not exist.
4.When I tried to make an OTU map, I received the following message:" Warning:OTU table appears to be using relative abudances and num_otu_hits was set to 5. Setting num_otu_hits to 0". What does it mean?
Thank you for your help!!

Hi there,
When you are working with a single sample in QIIME without a mapping file, the reads within the file will be considered as an individual sample. A mapping file with bar code is required to sort reads in your *.fa or *.sff file to respective sample. As its obvious that a single sample without multiplex run does not need to be split using split_library.py. But you need to include a mapping file with barcode sequence (you can use a string or sequence (generally 6 mer or 12 mer) at the start of all the sequences in the file. Refer this string of sequence as your bar code sequence in the mapping file against your sample id. Remember there is certain standard format for generating a mapping file which includes, #SampleID, BarcodeSequence, LinkerPrimerSequence and Description. The Linker primer sequence can be kept empty in the mapping file hoping your reads were prepossessed for quality and primer sequence. When running the split_library.py you have to bypass the LinkerPrimerSequence by providing with an optional command (i think its -p) in this split_library.py command (take help from qiime tutorial) otherwise the command wont be executed. Following this a file will be generated which removes the barcode sequence from all your reads in the file and assign them to be coming from a single sample.

The Biom file that you intend to work with works only with conjunction to the mapping_file so you have to provide a mapping file. Otherwise you can only generate the phylogenetic tree file and wont proceed further.

I have personally checked this method to work with Sanger sequenced data of 1kb to 1.5 kb and it works. Hope this helps you.

[trexbob]

Hi I can try to help... but you'll get better answers on the QIIME google web group.
Like rhinoceros says, you should really go over the tutorials, they answer a lot of questions a new person might have.
1) You can still use the split_libraries script to remove your primer/adapters/linkers and even reverse primers. I would suggest you at least try to remove your primer and adapter sequences as they will interfere with your analysis. You will need a mapping file for this, there is a page how to set that up. http://qiime.org/documentation/file_...-mapping-files
2) If you use a reference database (in lieu of de novo OTU picking) you can skip a lot of steps... again, take a look at the tutorial...
3) I believe the print_biom_table_summary was replaced by something else and is no longer in the current version of qiime. Do a search on the qiime google forum, and you should be able to find what happened to that command.
4) Sounds like it wants you to set a parameter differently, but I can't really help you as I don't know what exactly you were trying to do.

[jimmybee]

Just read the instructions on that page:
"Due spam on the QIIME Forum, we're requiring users to register before posting. As before, viewing the forum does not require registration. We're sorry for the inconvenience! Go here to register to post on the forums.

[rhinoceros]

Why don't you go over the qiime tutorials to get a general idea of how it works?

[litali]

qiime forum

I found how to post there, thank you

[litali]

qiime forum

I tried to use the qiime forum, but I son't see where can I post a new message there,,,

[jimmybee]

May I suggest emailing the Qiime forum (https://groups.google.com/forum/#!forum/qiime-forum). They're usually very quick in responding and are probably infinitely more skilled in this subject area

[litali]

I have 0ne 16s sample (without barcodes, as this is only one sample) sequenced by 454. I want to use qiime to analyze it:
1. Which step do I need to start from ? It is only one sample so do I need the steps of preparing the mapping file and the "assign sample to multiplex reads"?
If I do need them, how to do this in case this is only one unbarcoded sample? If I do not need it, and begin from the "pick OTU" step, I will lose the "filtering step" which is part of the " assign samples to multiplex reads", won't I?
2.Can I skip the "align OTU sequences" and the "filter alignment" and "phylogenetic tree" if all I am interested in is the OTUS whixh were found in the sample and the abunduncies in this only sample?
3.I made an OTU table, but when I tried to view the statistics of it via the command :" print_biom_table_smmary.py" I received a message that the command does not exist.
4.When I tried to make an OTU map, I received the following message:" Warning:OTU table appears to be using relative abudances and num_otu_hits was set to 5. Setting num_otu_hits to 0". What does it mean?
Thank you for your help!!