AcetoScan

• Version: 1.0
• Last modified: Thu Sep 3 12:52:30 CEST 2020
• Sign: Abhijeet Singh (abhijeetsingh.aau@gmail.com)

Description

AcetoScan is a software pipeline for the analysis of Illumina MiSeq sequencing data for the FTHFS (Formyl--tetrahydrofolate synthetase) gene/amplicons

AcetoScan can also process fasta sequences to filter out non-target sequences, assigning taxonomy to the FTHFS fasta sequences and generate phylogenetic tree (see AcetoScan commands)

Dependencies

AcetoScan pipeline uses some software dependencies (version equals or higher)

	- Cutadapt 	(2.9)
	- VSEARCH 	(2.13.1)
	- NCBI-blast+ 	(2.5.0+)
	- Bioperl 	(1.7.2-3)
	- MAFFT		(7.307)
	- Fasttree	(2.1.9)
	- R 		(3.5.2), with libraries:
		¤ phyloseq 	(1.24.2)
		¤ ggplot2 	(3.1.1)
		¤ plotly 	(4.9.0)
		¤ RColorBrewer 	(1.1.2)
		¤ plyr 		(1.8.4)
		¤ dplyr 	(0.8.0.1)
		¤ vegan		(2.5.6)

Installation

For installation run the following command in terminal, this will check all dependencies and download the reference database from acetobase website.

$ chmod +x install_linux.sh OR install_mac.sh

$ sudo ./install_linux.sh OR install_mac.sh

Installation without sudo/ROOT

For installation as local user make sure the dependency software are installed and modules are loaded (on server environment)

bash install_linux.sh OR install_mac.sh

acetoscan installation

install_linux.sh OR install_mac.sh will create main directory acetoscan in $HOME and sub-directories

1. bin - containing all main AcetoScan command scripts
2. dat - containing test
3. db - containing reference database AcetoBase
4. doc - containing user manual and tutorial video
5. scripts - containing dependencies scripts

AcetoScan input

1. user_data_directory - containing input raw data

Raw data must in format "Samplename_XYZ_L001_R1_001.fastq.(gz or bz2)"

https://support.illumina.com/content/dam/illumina-support/documents/documentation/software_documentation/miseqreporter/miseq-reporter-generate-fastq-workflow-guide-15042322-01.pdf, page 9, FASTQ File Names

AcetoScan output

acetoscan will result in two directories

• Directories will be created in default/destination path

1. output_data - containing process data will be generated and stored. In case of process failure, data can be accessed from here for further processing

2. acetoscan_result - containing all the final graphics, OTU table and TAX table. After successful execution of analysis, all the important data will be copies to this final directory.

AcetoScan commands

$ acetoscan -X

or

$ /$HOME/acetoscan/acetoscan -X

1. acetoscan       - for complete processing of raw sequence data
2. acetocheck      - for processing fasta sequences and filter out non-target sequences
3. acetotax        - acetocheck + taxonomic assignments
4. acetotree       - acetotax + phylogenetic tree generation

Using acetoscan program

Use acetoscan as follows

acetoscan -i /<input path>/ [-o /<output path>/] [-m 300] [-n 120] [-q 20] [-l 24] [-r 1] [-t 0.80] [-c 2] [-e 1e-3] [-B 1000] [-P 8]


        -i      Input directory containing raw illumina data
        -o      Output directory
                        :default = /$HOME/acetoscan/output_data
        -m      Maximum length of sequence after quality filtering
                        :default max_length = 300
        -n      Minimum length of sequence after quality filtering
                        :default min_length = 120
        -q      Quality threshold for the sequences
                        :default quality threshold = 20
        -l      Primer length
                        :default primer length = 24
        -r      Read type either forward or reverse reads
                        1 = forward reads (default), 2 = reverse reads
        -t      Clustering threshold
                        :default cluster threshold = 0.80 (80 %)
        -c      Minimum cluster size
                        :default minimum cluster size = 2
        -e      E-value
                        :default evalue = 1e-3
        -B      Bootstrap value
                        :default bootstrap = 1000
        -P      Parallel processes / threads
                        :default no. of parallels = all available threads
        -h      Print help
        -X      Print AcetoScan commands
        -v      Print AcetoScan version
        -C      Print AcetoScan citation

acetocheck

Use this command for the FTHFS fasta a sequences and filter out any unspecific / non-FTHFS sequence

$ acetocheck -h

acetocheck -i /path/<input_file> [-o /path/<output_file>] [-e 1e-3] [-P 8]


        -i      Input file - multifasta file
        -o      Output file
                        :default = acetocheck_<date>_<time>.fasta
        -e      E-value
                        :default evalue = 1e-3
        -P      Parallel processes/threads
                        :default no. of parallels = all available threads
        -h      Print help
        -X      Print AcetoScan commands
        -v      Print AcetoScan version
        -C      Print AcetoScan citation

Use

acetocheck -i /home/abhi/Desktop/seq.fasta -o /home/abhi/Desktop/my_sequences -e 1e-3 -P 8

output

1. my_sequences.fasta
2. acetotax_< Date >_< Time >.log

acetotax

Use this command for the FTHFS fasta a sequences and filter out any unspecific / non-FTHFS sequence and taxonomic annotations of the fasta sequences

$ acetotax -h

acetotax -i /path/<input_file>/ [-o /path/<output_file>/] [-e 1e-3] [-P 8]

        -i      Input_file
        -o      Output file
                        :default = acetotax_<date>_<time>.csv
                        :default = acetotax_<date>_<time>.fasta
        -e      E-value
                        :default evalue = 1e-3
        -P      Parallel processes/threads
                        :default no. of parallels = all available threads
        -h      Print help
        -X      Print AcetoScan commands
        -v      Print AcetoScan version
        -C      Print AcetoScan citation

Use

acetotax -i /home/abhi/Desktop/seq.fasta -o /home/abhi/Desktop/my_sequences -e 1e-3 -P 8

output

1. my_sequences.fasta
2. my_sequences.csv
3. acetotax_< Date >_< Time >.log

acetotree

Use this command for the FTHFS fasta a sequences and filter out any unspecific / non-FTHFS sequence and taxonomic annotations of the fasta sequences and generation of phylogenetic tree

$ acetotree -h

acetotree -i /path/<input_file> [-o /path/<output_file>] [-e 1e-3] [-B 1000] [-P 8]


        -i      Input file - multifasta file
        -o      Output file
                        :default = acetotree_<date>_<time>.fasta
                        :default = acetotree_<date>_<time>.csv
                        :default = acetotree_<date>_<time>.aln
                        :default = acetotree_<date>_<time>.tree
        -e      E-value
                        :default evalue = 1e-3
        -B      Bootstrap value
                        :default bootstrap = 1000
        -P      Parallel processes/threads
                        :default no. of parallels = all available threads
        -h      Print help
        -X      Print AcetoScan commands
        -v      Print AcetoScan version
        -C      Print AcetoScan citation

Use

acetotree -i /home/abhi/Desktop/seq.fasta -o /home/abhi/Desktop/my_sequences -e 1e-3 -B 1000 -P 8

output

1. my_sequences.fasta
2. my_sequences.csv
3. my_sequences.aln
4. my_sequences.tree
5. acetotax_< Date >_< Time >.log

Running AcetoScan Pipelline as a Docker image/container

Mounting the local volume / connecting the files on local computer to the container

sudo docker volume create --opt type=none --opt o=bind --opt device=/PATH/to/my/DATA --name MY_CUSTOM_NAME

Example

Here

--opt device= - will have the path to your raw input data

--name - any name according to your wish

sudo docker volume create --opt type=none --opt o=bind --opt device=/home/abhi/Desktop/reads --name myDockerAcetoscan

Running docker image as container

NOTE: only MY_CUSTOM_NAME should be changed according to your --name flag

sudo docker run --rm -v MY_CUSTOM_NAME:/acetoscan/input_dir -it abhijeetsingh1704/acetoscan -i /acetoscan/input_dir

Example

sudo docker run --rm -v myDockerAcetoscan:/acetoscan/input_dir -it abhijeetsingh1704/acetoscan -i /acetoscan/input_dir

Default program for AcetoScan pipeline is acetoscan command, therefore it is optional to call it with --entrypoint flag. But in case of acetocheck,acetotax or acetotree command the code need to include --entrypoint flag

Example

acetoscan

sudo docker run --rm -v myDockerAcetoscan:/acetoscan/input_dir --entrypoint acetoscan -it abhijeetsingh1704/acetoscan -i /acetoscan/input_dir

acetocheck

sudo docker run --rm -v myDockerAcetoscan:/acetoscan/input_dir --entrypoint acetocheck -it abhijeetsingh1704/acetoscan -i /acetoscan/input_dir/input_file.fasta

• OR

sudo docker run --rm -v myDockerAcetoscan:/acetoscan/input_dir --entrypoint acetocheck -it abhijeetsingh1704/acetoscan -i /acetoscan/input_dir/input_file.fasta -o /acetoscan/input_dir/output_file.fasta

Citation

Singh A, Nylander JAA, Schnürer A, Bongcam-Rudloff E and Müller B (2020) High-Throughput Sequencing and Unsupervised Analysis of Formyltetrahydrofolate Synthetase (FTHFS) Gene Amplicons to Estimate Acetogenic Community Structure. Front. Microbiol. 11:2066. doi: 10.3389/fmicb.2020.02066