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Update pipeline doc to GL-DPPD-7104-D #202

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47 changes: 35 additions & 12 deletions Amplicon/Illumina/Pipeline_GL-DPPD-7104_Versions/GL-DPPD-7104-C.md
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Original file line number Diff line number Diff line change
Expand Up @@ -45,15 +45,15 @@ Software Updates and Changes:
| hexbin | N/A | 1.28.3 |
| mia | N/A | 1.14.0 |
| phyloseq | N/A | 1.50.0 |
| rcolorbrewer | N/A | 1.1.3 |
| RColorBrewer | N/A | 1.1.3 |
| taxize | N/A | 0.10.0 |
| tidyverse | N/A | 2.0.0 |
| vegan | N/A | 2.6-10 |
| vsn | N/A | 3.74.0 |
| patchwork | N/A | 1.3.0 |
| rstatix | N/A | 0.7.2 |
| multcompView | N/A | 0.1-10 |
| scales | N/A | 1.4.0 |
| scales | N/A | 1.3.0 |
| dendextend | N/A | 1.19.0 |

- Added new processing steps in R to generate processed data outputs for alpha and beta diversity, taxonomic summary plots, and differential abundance:
Expand All @@ -63,7 +63,7 @@ Software Updates and Changes:
- Differential Abundance Testing ([Step 10](#9-differential-abundance-analysis)) with
ANCOMBC 1 ([Step 10a](#10a-ancombc-1)), ANCOMBC 2 ([Step 10b](#10b-ancombc-2)), and Deseq2 ([Step 10c](#10c-deseq2))
- Assay-specific suffixes were added where needed for OSDR ("_GLAmpSeq")
- Updated [DECIPHER](https://www2.decipher.codes/data/Downloads/TrainingSets/) reference files to the following:
- Updated [DECIPHER](https://decipher.codes/Downloads.html) reference files to the following:
- ITS UNITE: "UNITE\_v2024\_April2024.RData"
- SILVA SSU r138: "SILVA\_SSU\_r138\_2\_2024.RData"
- PR2 v4.13: "PR2\_v4\_13\_March2021.RData"
Expand Down Expand Up @@ -128,6 +128,7 @@ Software Updates and Changes:
|DADA2|1.34.0|[https://www.bioconductor.org/packages/release/bioc/html/dada2.html](https://www.bioconductor.org/packages/release/bioc/html/dada2.html)|
|DECIPHER|3.2.0|[https://bioconductor.org/packages/release/bioc/html/DECIPHER.html](https://bioconductor.org/packages/release/bioc/html/DECIPHER.html)|
|biomformat|1.34.0|[https://github.com/joey711/biomformat](https://github.com/joey711/biomformat)|
|dp_tools|1.3.8|[https://github.com/torres-alexis/dp_tools](https://github.com/torres-alexis/dp_tools)|
|ANCOMBC|2.8.0|[https://github.com/FrederickHuangLin/ANCOMBC](https://github.com/FrederickHuangLin/ANCOMBC)|
|broom|1.0.7|[https://CRAN.R-project.org/package=broom](https://CRAN.R-project.org/package=broom)|
|DescTools|0.99.59|[https://andrisignorell.github.io/DescTools/](https://andrisignorell.github.io/DescTools/)|
Expand All @@ -139,15 +140,15 @@ Software Updates and Changes:
|hexbin|1.28.3|[https://CRAN.R-project.org/package=hexbin](https://CRAN.R-project.org/package=hexbin)|
|mia|1.14.0|[https://github.com/microbiome/mia](https://github.com/microbiome/mia)|
|phyloseq|1.50.0|[https://bioconductor.org/packages/release/bioc/html/phyloseq.html](https://bioconductor.org/packages/release/bioc/html/phyloseq.html)|
|rcolorbrewer|1.1.3|[https://CRAN.R-project.org/package=RColorBrewer](https://CRAN.R-project.org/package=RColorBrewer)|
|RColorBrewer|1.1.3|[https://CRAN.R-project.org/package=RColorBrewer](https://CRAN.R-project.org/package=RColorBrewer)|
|taxize|0.10.0|[https://docs.ropensci.org/taxize/](https://docs.ropensci.org/taxize/)|
|tidyverse|2.0.0|[https://CRAN.R-project.org/package=tidyverse](https://CRAN.R-project.org/package=tidyverse)|
|vegan|2.6-10|[https://cran.r-project.org/package=vegan](https://cran.r-project.org/package=vegan)|
|vsn|3.74.0|[https://bioconductor.org/packages/release/bioc/html/vsn.html](https://bioconductor.org/packages/release/bioc/html/vsn.html)|
|patchwork|1.3.0|[https://CRAN.R-project.org/package=patchwork](https://CRAN.R-project.org/package=patchwork)|
|rstatix|0.7.2|[https://CRAN.R-project.org/package=rstatix](https://CRAN.R-project.org/package=rstatix)|
|multcompView|0.1-10|[https://CRAN.R-project.org/package=multcompView](https://CRAN.R-project.org/package=multcompView)|
|scales|1.4.0|[https://CRAN.R-project.org/package=scales](https://CRAN.R-project.org/package=scales)|
|scales|1.3.0|[https://CRAN.R-project.org/package=scales](https://CRAN.R-project.org/package=scales)|
|dendextend|1.19.0|[https://CRAN.R-project.org/package=dendextend](https://CRAN.R-project.org/package=dendextend)|

# Reference databases used
Expand Down Expand Up @@ -205,13 +206,18 @@ multiqc --interactive -n raw_multiqc_GLAmpSeq -o /path/to/raw_multiqc/output/raw
zip -r raw_multiqc_GLAmpSeq_report.zip raw_multiqc_GLAmpSeq_report
```

**Parameter Definitions:**

**Parameter Definitions:**
**multiqc**
- `--interactive` – force reports to use interactive plots
- `-n` – prefix name for output files
- `-o` – the output directory to store results
- `/path/to/directory/containing/raw_fastqc/files` – the directory holding the output data from the FastQC run, provided as a positional argument

**zip**
- `-r` - recurse into directories
- `raw_multiqc_GLAmpSeq_report.zip` – positional argument naming the zip output file
- `raw_multiqc_GLAmpSeq_report` – positional argument naming the input folder to package

**Input Data:**

* \*fastqc.zip (FastQC output data, output from [Step 1a](#1a-raw-data-qc))
Expand Down Expand Up @@ -347,13 +353,18 @@ multiqc --interactive -n filtered_multiqc_GLAmpSeq -o /path/to/filtered_multiqc/
zip -r filtered_multiqc_GLAmpSeq_report.zip filtered_multiqc_GLAmpSeq_report
```

**Parameter Definitions:**

**Parameter Definitions:**
**multiqc**
- `--interactive` – force reports to use interactive plots
- `-n` – prefix name for output files
- `-o` – the output directory to store results
- `/path/to/directory/containing/filtered_fastqc/files` – the directory holding the output data from the FastQC run, provided as a positional argument

**zip**
- `-r` - recurse into directories
- `filtered_multiqc_GLAmpSeq_report.zip` – positional argument naming the zip output file
- `filtered_multiqc_GLAmpSeq_report` – positional argument naming the input folder to package

**Input Data:**

* \*fastqc.zip (FastQC output data, output from [Step 4a](#4a-filtered-data-qc))
Expand All @@ -371,7 +382,7 @@ zip -r filtered_multiqc_GLAmpSeq_report.zip filtered_multiqc_GLAmpSeq_report
## 5. Calculate Error Mdel, Apply DADA2 Algorithm, Assign Taxonomy, and Create Output Tables
> The following is run in an R environment.

These example commands as written assume paired-end data, with notes included on what would be different if working with single-end data. The taxonomy reference database used below is an example only, suitable for the example 16S dataset ([GLDS-200](https://osdr.nasa.gov/bio/repo/data/studies/OSD-200)) used here. Other taxonomy references databases designed for DECIPHER can be found here: [https://www2.decipher.codes/data/Downloads/TrainingSets/](https://www2.decipher.codes/data/Downloads/TrainingSets/)
These example commands as written assume paired-end data, with notes included on what would be different if working with single-end data. The taxonomy reference database used below is an example only, suitable for the example 16S dataset ([GLDS-200](https://osdr.nasa.gov/bio/repo/data/studies/OSD-200)) used here. Other taxonomy references databases designed for DECIPHER can be found here: [https://decipher.codes/Downloads.html](https://decipher.codes/Downloads.html)

<br>

Expand Down Expand Up @@ -507,7 +518,7 @@ seqtab.nochim <- removeBimeraDenovo(unqs=seqtab, method="consensus", multithread
dna <- DNAStringSet(getSequences(seqtab.nochim))

## Downloading the reference R taxonomy object: ##
download.file(url = "https://figshare.com/ndownloader/files/52846199",
download.file(url = "https://api.figshare.com/v2/file/download/52846199",
destfile = "SILVA_SSU_r138_2_2024.RData",
method = "libcurl",
headers = c("User-Agent" = "Mozilla/5.0"))
Expand All @@ -517,6 +528,7 @@ load("SILVA_SSU_r138_2_2024.RData")

## Classifying sequences:
tax_info <- IdTaxa(test=dna, trainingSet=trainingSet, strand="both", processors=NULL)
```

**Parameter Definitions:**

Expand Down Expand Up @@ -584,6 +596,16 @@ write_biom(biom_object, "taxonomy-and-counts_GLAmpSeq.biom")
tax_and_count_tab <- merge(tax_tab, asv_tab)
write.table(tax_and_count_tab, "taxonomy-and-counts_GLAmpSeq.tsv", sep="\t", quote=FALSE, row.names=FALSE)
```
```bash
zip -j -q taxonomy-and-counts_GLAmpSeq.biom.zip taxonomy-and-counts_GLAmpSeq.biom
```

**Parameter Definitions:**
**zip**
- `-j` - junk (don't record) directory names
- `-q` – quiet operation
- `taxonomy-and-counts_GLAmpSeq.biom.zip` – positional argument naming the zip output file
- `taxonomy-and-counts_GLAmpSeq.biom` – positional argument naming the input folder to package

**Input Data:**

Expand All @@ -596,7 +618,8 @@ write.table(tax_and_count_tab, "taxonomy-and-counts_GLAmpSeq.tsv", sep="\t", quo
* **counts_GLAmpSeq.tsv** (a tab-separated file containing the sample feature count table)
* **taxonomy_GLAmpSeq.tsv** (a tab-separated file containing the taxonomy table)
* **taxonomy-and-counts_GLAmpSeq.tsv** (a tab-separated file containing the combined taxonomy and count table)
* **taxonomy-and-counts_GLAmpSeq.biom** (a biom-formatted file containing the count and taxonomy table)
* **taxonomy-and-counts_GLAmpSeq.biom.zip** (a zip package containing the biom-formatted file)
* taxonomy-and-counts_GLAmpSeq.biom (a biom-formatted file containing the count and taxonomy table)
* **read-count-tracking_GLAmpSeq.tsv** (a tab-separated file containing the read counts at each processing step)

<br>
Expand Down
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