I am completely new in this QIIME's world.
I have been trying to analyze my data set, it is composed by 2 samples:
Sample 1: V1V2, V3V4, V4V6 16S rRNA amplicon.
Sample 2: V3V4 16
So, the data is demultiplexed, I have fastq files like this:
@M02169:22:000000000-AAWG6:1:1101:14717:1713 1:N:0:TAGGCCTG+TCTCTCCG
CCTACGGGTGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGA----------Sequence--------
I have two reads for each samples:
Sample_S79_L001_R1_001.fastq
Sample_S79_L001_R2_001.fastq
Also, I performed a merge of this sequences using PEAR, and I got the the assembled sequences.
I´ve been following several tutorials found in forums, but I still don´t understand how to process this data.
In addition, I do not know how to get the mapping file because of the structure of the sequences provided by MiSeq.
I would really appreciate any help.
If you are working with different regions of 16S rRNA you have to take only one kind of amplicon for the data analysis, and you can do it without maping file.
You can split all your samples data:
multiple_split_libraries_fastq.py -i input_directory -o output_directory
and use some chimera detector, like vsearch:
vsearch --uchime_ref ../seqs.fna --db gold.fa --nonchimeras seqs.nonchimeras.fna --threads 6
And after that do the OTU picking:
pick_open_reference_otus.py -o otus/ -i split/seqs.nonchimeras.fna
And you obtain the OTUs tables for diversity and taxonomical analysis.
Good luck!!
If you are working with different regions of 16S rRNA you have to take only one kind of amplicon for the data analysis, and you can do it without maping file.
You can split all your samples data:
multiple_split_libraries_fastq.py -i input_directory -o output_directory
and use some chimera detector, like vsearch:
vsearch --uchime_ref ../seqs.fna --db gold.fa --nonchimeras seqs.nonchimeras.fna --threads 6
And after that do the OTU picking:
pick_open_reference_otus.py -o otus/ -i split/seqs.nonchimeras.fna
And you obtain the OTUs tables for diversity and taxonomical analysis.
Good luck!!
Dear Jose and Jesus,
I was following your conversations just wanted to know if we have % of species from 16s sequencing, what are questions we can ask? Can we use this data as other data like gene expression? or data need to be analysed a bit different way?
Thanks
Hi Manuel!
- I suggest this guide: http://www.earthmicrobiome.org/protocols-and-standards/initial-qiime-processing/
- Also, for a better outlook see this publication: https://www.researchgate.net/publication/322266856_Characterization_of_the_Skin_Microbiota_of_the_Cane_Toad_Rhinella_cf_marina_in_Puerto_Rico_and_Costa_Rica
I hope it helps,
Gilmary Ortiz
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