population-diversity:microbiome-analysis-with-qiime2-using-illumina-paired-end-sequence-data
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population-diversity:microbiome-analysis-with-qiime2-using-illumina-paired-end-sequence-data [2020/03/28 11:07] – bngina | population-diversity:microbiome-analysis-with-qiime2-using-illumina-paired-end-sequence-data [2020/04/29 15:02] – [Qiime2 data filtering and feature (OTU) table construction] bngina | ||
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==== Importing data into Qiime2 ==== | ==== Importing data into Qiime2 ==== | ||
- | [[https:// | + | [[https:// |
+ | |||
+ | We import raw fastq files into qiime2. Depending on the sequencing platform used to generate the data, you will have data that has been de-multiplexed into sample specif fastq files, or not. The MiSeq platform at [[ https:// | ||
+ | |||
+ | Read more about [[ https:// | ||
+ | |||
+ | To import fastq files; | ||
+ | |||
+ | < | ||
+ | |||
+ | #i create a directory to store all my artifact i.e ' | ||
+ | |||
+ | mkdir / | ||
+ | |||
+ | #import the fastq files | ||
+ | |||
+ | qiime tools import \ | ||
+ | | ||
+ | | ||
+ | | ||
+ | | ||
+ | </ | ||
+ | |||
+ | We mentioned that qiime2 provides method to summarize and view artifacts by storing them as visualizations, | ||
+ | |||
+ | < | ||
+ | |||
+ | qiime demux summarize \ | ||
+ | | ||
+ | | ||
+ | </ | ||
+ | |||
+ | ==== Qiime2 data filtering and feature (OTU) table construction ==== | ||
+ | |||
+ | Qiime2 provides two pluggins/ | ||
+ | |||
+ | Important to keep in mind is the size of the amplified region covered by the primers used and what is the expected fragment length after read joining? This will determine the trim and truncate length parameters for dada2 and deblur pipelines. | ||
+ | |||
+ | === Dada2 === | ||
+ | |||
+ | The Dada2 pipeline detects and corrects illumina amplicon sequence data and additionally filters any phiX reads identified in sequencing data and also filter out any chimeric sequences. | ||
+ | To see usage and parameters that can be adjusted get the help for the pluggin i.e | ||
+ | |||
+ | < | ||
+ | qiime dada2 --help | ||
+ | </ | ||
+ | |||
+ | The data we are using is paired-end, hence we will use the [[https:// | ||
+ | |||
+ | A key parameter to be careful about is the //'' | ||
+ | |||
+ | The other key parameters in quality control of the sequences are those used to trim the forward, '' | ||
+ | |||
+ | To determine what values to pass for these two parameters; | ||
+ | *Review the Interactive Quality Plot tab in the //'' | ||
+ | *The expected fragment size after the reads are joined, particularly for the //'' | ||
+ | |||
+ | The command would therefore be | ||
+ | |||
+ | < | ||
+ | qiime dada2 denoise-paired \ | ||
+ | | ||
+ | | ||
+ | | ||
+ | | ||
+ | | ||
+ | | ||
+ | | ||
+ | | ||
+ | | ||
+ | |||
+ | #next summarise the features table and view, | ||
+ | |||
+ | time qiime feature-table summarize \ | ||
+ | | ||
+ | | ||
+ | |||
+ | |||
+ | #view a summary of the rep sequences | ||
+ | |||
+ | time qiime feature-table tabulate-seqs \ | ||
+ | | ||
+ | | ||
+ | |||
+ | #and also view the denoising statistics | ||
+ | time qiime metadata tabulate \ | ||
+ | --m-input-file ${data_dir}/ | ||
+ | --o-visualization ${qzv_dir}/ | ||
+ | </ |
population-diversity/microbiome-analysis-with-qiime2-using-illumina-paired-end-sequence-data.txt · Last modified: 2020/04/29 15:09 by bngina