Amplicon Sequence Analysis Pipeline (ASAP 1.4, May 2018)
This is an automated pipeline for analysis of amplicon sequence data (could be multiple sequencing runs) including 16S, 18S and ITS.
It wraps QIIME commands and complements them with additional analysis where QIIME is not good at, such as combine multiple sequencing runs, OTU clustering and chimeric removal with UPARSE, alignment filtering with Gblock, removing Chloroplast sequences.
The results include OTU sequences, OTU table, taxonomic classification, community composition charts, PCoA charts (UniFrac, Bray Curtis ...), Alpha diversity indexes (OTU number, Shannon, Chao1, Goods Coverage, PD_whole, Simposon ...), rarefaction curve, and processed Fastq files for submission to NCBI.
Below is the work flow:
- Search datasets (It accepts multiple sequencing runs)
- Read quality check - FastQC
- Merge pair-end sequence files - PEAR
- Demultiplexing (split library) - QIIME (1.9.1)
- Trim forward and reverse primer and any extra bases
- Merge sequence of different datasets
- Merge map files
- Dereplicate reads and calculate abundance - VSEARCH
- OTU clustering, chimera and singleton removal - UPARSE
- Make OTU table - VSEARCH
- Taxonomic classification of OTU - RDP Classifier
- Remove Chloroplast and Mitochondria from OTU table
- Alignment and filtering - MAFFT & Gblock
- Phylogenetic tree construction - QIIME
- Convert and resample OTU table - QIIME
- Read summary
- Make taxonomic summary chart - QIIME
- Make heat map (phylum to family) - R script
- Alpha diversity - QIIME
- Beta diversity and plots - QIIME
- Summary
For more information, see the slides. To analyze your data, go to Job Submission. Click here for user log.