issues with minion_mobile_lab

[asogg]

Hello Simone, I installed the program on Ubuntu 20.04.3 LTS following the instructions and I tried initially to create the database with import_database.sh but using ncbi the process is killed, perhaps a RAM problem. so I used silva and I was able to create the .qza files. At the time of using the script Launch_MinION_mobile_lab.sh (which I attach) the process gives errors at the time of doing the quality control (I attach the file nohup.out). What can be the problem (or problems). grazie!

Launch_MinION_mobile_lab.sh !/bin/bash

#

Copyright 2021 Simone Maestri. All rights reserved.

Simone Maestri simone.maestri@univr.it

#

This program is free software: you can redistribute it and/or modify

it under the terms of the GNU General Public License as published by

the Free Software Foundation, either version 3 of the License, or

(at your option) any later version.

#

This program is distributed in the hope that it will be useful,

but WITHOUT ANY WARRANTY; without even the implied warranty of

MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the

GNU General Public License for more details.

#

You should have received a copy of the GNU General Public License

along with this program. If not, see http://www.gnu.org/licenses/.

#

RAW_READS_DIR=fast5_pass RAW_READS_DIR_FULL=$(./fast5_pass $RAW_READS_DIR) source activate MetONTIIME_env PIPELINE_DIR=$( /home/as/MetONTIIME $( dirname "${BASH_SOURCE[0]}" )) nohup Rscript /home/as/MetONTIIME/MinION_mobile_lab.R /home/as/MetONTIIME/config_MinION_mobile_lab.R /home/as/MetONTIIME/fast5_pass

nohup.out Welcome to MinION meta-barcoding mobile laboratory!

Raw reads directory: /home/as/MetONTIIME/fast5_pass Basecalled reads directory: /home/as/MetONTIIME/fast5_pass_analysis/basecalling Preprocessing directory: /home/as/MetONTIIME/fast5_pass_analysis/preprocessing Analysis and results directory: /home/as/MetONTIIME/fast5_pass_analysis/analysis Flow-cell: FLO-MIN106 Kit: SQK-RAB204 Expected amplicon length [bp]: 1400 Barcodes used in this experiment: BC01, BC02, BC03, BC04, BC05, BC06, BC07, BC08, BC09, BC10, BC11, BC12 Basecalling is going to be performed by : Guppy Basecalling Software, (C) Oxford Nanopore Technologies, Limited. Version 5.0.16+b9fcd7b Basecalling model: fast Demultiplexing is going to be performed by guppy_barcoder after basecalling Taxonomic classifier: Vsearch

Basecalling started at Wed Nov 17 21:48:38 2021 ONT Guppy basecalling software version 5.0.16+b9fcd7b config file: /home/as/ont-guppy-cpu/data/dna_r9.4.1_450bps_fast.cfg model file: /home/as/ont-guppy-cpu/data/template_r9.4.1_450bps_fast.jsn input path: /home/as/MetONTIIME/fast5_pass save path: /home/as/MetONTIIME/fast5_pass_analysis/basecalling chunk size: 2000 chunks per runner: 160 minimum qscore: 8 records per file: 4000 num basecallers: 4 cpu mode: ON threads per caller: 8 Found 10 fast5 files to process. Init time: 60 ms

0% 10 20 30 40 50 60 70 80 90 100% |----|----|----|----|----|----|----|----|----|----|

---

Caller time: 91887 ms, Samples called: 12953296, samples/s: 140970 Finishing up any open output files. Basecalling completed successfully. Basecalling finished at Wed Nov 17 21:50:10 2021

Demultiplexing started at Wed Nov 17 21:50:10 2021

ONT Guppy barcoding software version 5.0.16+b9fcd7b input path: /home/as/MetONTIIME/fast5_pass_analysis/basecalling save path: /home/as/MetONTIIME/fast5_pass_analysis/preprocessing arrangement files: barcode_arrs_rab.cfg lamp arr. files: barcode_arrs_ncov8.cfg barcode_arrs_ncov96.cfg barcode_arrs_multivirus1.cfg barcode_arrs_multivirus8.cfg min. score front: 60 min. score rear: 60

Found 2 input files.

0% 10 20 30 40 50 60 70 80 90 100% |----|----|----|----|----|----|----|----|----|----|

---

Done in 264 ms. Demultiplexing finished at Wed Nov 17 21:50:10 2021

Number of basecalled reads: 1035

Creating folder: /home/as/MetONTIIME/fast5_pass_analysis/qc, which is going to include stats about the sequencing run

Now performing quality control with PycoQC

sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/pycoQC: not found sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found Mean read length (stdev) for sample BC01: NaN (NA) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC01 sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/NanoFilt: not found sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found WARNING: skipping sample BC01, since no reads survived the length or quality filtering!

sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found Mean read length (stdev) for sample BC02: NaN (NA) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC02 sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/NanoFilt: not found sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found WARNING: skipping sample BC02, since no reads survived the length or quality filtering!

sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found Mean read length (stdev) for sample BC03: NaN (NA) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC03 sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/NanoFilt: not found sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found WARNING: skipping sample BC03, since no reads survived the length or quality filtering!

sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found Mean read length (stdev) for sample BC04: NaN (NA) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC04 sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/NanoFilt: not found sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found WARNING: skipping sample BC04, since no reads survived the length or quality filtering!

sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found Mean read length (stdev) for sample BC07: NaN (NA) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC07 sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/NanoFilt: not found sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found WARNING: skipping sample BC07, since no reads survived the length or quality filtering!

sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found Mean read length (stdev) for sample BC08: NaN (NA) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC08 sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/NanoFilt: not found sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found WARNING: skipping sample BC08, since no reads survived the length or quality filtering!

sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found Mean read length (stdev) for sample BC09: NaN (NA) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC09 sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/NanoFilt: not found sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found WARNING: skipping sample BC09, since no reads survived the length or quality filtering!

sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found Mean read length (stdev) for sample BC10: NaN (NA) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC10 sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/NanoFilt: not found sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found WARNING: skipping sample BC10, since no reads survived the length or quality filtering!

sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found Mean read length (stdev) for sample BC11: NaN (NA) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC11 sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/NanoFilt: not found sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found WARNING: skipping sample BC11, since no reads survived the length or quality filtering!

There were 28 warnings (use warnings() to see them) Error in h(simpleError(msg, call)) : error in evaluating the argument 'x' in selecting a method for function 'grep': cannot open the connection Calls: grep -> readLines -> file -> .handleSimpleError -> h In addition: Warning messages: 1: In file(con, "r") : 'raw = FALSE' but '/home/as/MetONTIIME/fast5_pass_analysis/analysis/' is not a regular file 2: In file(con, "r") : cannot open file '/home/as/MetONTIIME/fast5_pass_analysis/analysis/': it is a directory Execution halted

[MaestSi]

Hi, the relevant errors are:

sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/pycoQC: not found
sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found
sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/NanoFilt: not found

So, basically, it looks like the creation of the MetONTIIME_env environment was not successful. Could you please check you can activate che environment with: source activate MetONTIIME_env and that pycoQC, seqtk and NanoFilt executables are in the path? Thanks, Simone

[asogg]

Hi Simone, the environment could be activated and in the environment command line the executables start and are also in the path, maybe something wrong in the path..see the end of .bashrc

~/.bashrc: executed by bash(1) for non-login shells.

see /usr/share/doc/bash/examples/startup-files (in the package bash-doc)

for examples

If not running interactively, don't do anything

case $- in i) ;; *) return;; esac

don't put duplicate lines or lines starting with space in the history.

See bash(1) for more options

HISTCONTROL=ignoreboth

append to the history file, don't overwrite it

shopt -s histappend

for setting history length see HISTSIZE and HISTFILESIZE in bash(1)

HISTSIZE=1000 HISTFILESIZE=2000

check the window size after each command and, if necessary,

update the values of LINES and COLUMNS.

shopt -s checkwinsize

If set, the pattern "**" used in a pathname expansion context will

match all files and zero or more directories and subdirectories.

shopt -s globstar

make less more friendly for non-text input files, see lesspipe(1)

[ -x /usr/bin/lesspipe ] && eval "$(SHELL=/bin/sh lesspipe)"

set variable identifying the chroot you work in (used in the prompt below)

if [ -z "${debian_chroot:-}" ] && [ -r /etc/debian_chroot ]; then debian_chroot=$(cat /etc/debian_chroot) fi

set a fancy prompt (non-color, unless we know we "want" color)

case "$TERM" in xterm-color|*-256color) color_prompt=yes;; esac

uncomment for a colored prompt, if the terminal has the capability; turned

off by default to not distract the user: the focus in a terminal window

should be on the output of commands, not on the prompt

force_color_prompt=yes

if [ -n "$force_color_prompt" ]; then if [ -x /usr/bin/tput ] && tput setaf 1 >&/dev/null; then

We have color support; assume it's compliant with Ecma-48

# (ISO/IEC-6429). (Lack of such support is extremely rare, and such
# a case would tend to support setf rather than setaf.)
color_prompt=yes
else
color_prompt=
fi

fi

if [ "$color_prompt" = yes ]; then PS1='${debian_chroot:+($debian_chroot)}[\033[01;32m]\u@\h[\033[00m]:[\033[01;34m]\w[\033[00m]\$ ' else PS1='${debian_chroot:+($debian_chroot)}\u@\h:\w\$ ' fi unset color_prompt force_color_prompt

If this is an xterm set the title to user@host:dir

case "$TERM" in xterm|rxvt) PS1="[\e]0;${debian_chroot:+($debian_chroot)}\u@\h: \w\a]$PS1" ;; *) ;; esac

enable color support of ls and also add handy aliases

if [ -x /usr/bin/dircolors ]; then test -r ~/.dircolors && eval "$(dircolors -b ~/.dircolors)" || eval "$(dircolors -b)" alias ls='ls --color=auto'

alias dir='dir --color=auto'

#alias vdir='vdir --color=auto'

alias grep='grep --color=auto'
alias fgrep='fgrep --color=auto'
alias egrep='egrep --color=auto'

fi

colored GCC warnings and errors

export GCC_COLORS='error=01;31:warning=01;35:note=01;36:caret=01;32:locus=01:quote=01'

some more ls aliases

alias ll='ls -alF' alias la='ls -A' alias l='ls -CF'

Add an "alert" alias for long running commands. Use like so:

sleep 10; alert

alias alert='notify-send --urgency=low -i "$([ $? = 0 ] && echo terminal || echo error)" "$(history|tail -n1|sed -e '\''s/^\s[0-9]+\s//;s/[;&|]\s*alert$//'\'')"'

Alias definitions.

You may want to put all your additions into a separate file like

~/.bash_aliases, instead of adding them here directly.

See /usr/share/doc/bash-doc/examples in the bash-doc package.

if [ -f ~/.bash_aliases ]; then . ~/.bash_aliases fi

enable programmable completion features (you don't need to enable

this, if it's already enabled in /etc/bash.bashrc and /etc/profile

sources /etc/bash.bashrc).

if ! shopt -oq posix; then if [ -f /usr/share/bash-completion/bash_completion ]; then . /usr/share/bash-completion/bash_completion elif [ -f /etc/bash_completion ]; then . /etc/bash_completion fi fi

Anaconda3

export PATH="/home/as/miniconda3/bin:$PATH"

export PATH=home/as/miniconda3/bin:$PATH export PATH=/home/as/miniconda3/envs/MetONTIIME_env/bin/pycoQC:$PATH export PATH=/home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk:$PATH export PATH=/home/as/miniconda3/envs/MetONTIIME_env/bin/NanoFilt:$PATH

thank you Alessio

[MaestSi]

Since the tools look like properly installed and working, then just try running the script again as reported in the README (directly run the Launch_MinION_mobile_lab.sh script, without activating the environment before), and let me know if it still gives some errors. Ciao, Simone

[asogg]

i launched the script without activation see below

@as-HP-Pavilion-dv6-Notebook-PC:~/MetONTIIME$ ./Launch_MinION_mobile_lab.sh ./Launch_MinION_mobile_lab.sh: line 22: ./fast5_pass: Is a directory ./Launch_MinION_mobile_lab.sh: line 24: /home/as/MetONTIIME: Is a directory nohup: ignoring input and appending output to 'nohup.out'

the script executed

!/bin/bash

#

Copyright 2021 Simone Maestri. All rights reserved.

Simone Maestri simone.maestri@univr.it

#

This program is free software: you can redistribute it and/or modify

it under the terms of the GNU General Public License as published by

the Free Software Foundation, either version 3 of the License, or

(at your option) any later version.

#

This program is distributed in the hope that it will be useful,

but WITHOUT ANY WARRANTY; without even the implied warranty of

MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the

GNU General Public License for more details.

#

You should have received a copy of the GNU General Public License

along with this program. If not, see http://www.gnu.org/licenses/.

#

RAW_READS_DIR=fast5_pass RAW_READS_DIR_FULL=$(./fast5_pass $RAW_READS_DIR) source activate MetONTIIME_env PIPELINE_DIR=$( /home/as/MetONTIIME $( dirname "${BASH_SOURCE[0]}" )) nohup Rscript /home/as/MetONTIIME/MinION_mobile_lab.R /home/as/MetONTIIME/config_MinION_mobile_lab.R /home/as/MetONTIIME/fast5_pass

nohup.out

Welcome to MinION meta-barcoding mobile laboratory!

Raw reads directory: /home/as/MetONTIIME/fast5_pass Basecalled reads directory: /home/as/MetONTIIME/fast5_pass_analysis/basecalling Preprocessing directory: /home/as/MetONTIIME/fast5_pass_analysis/preprocessing Analysis and results directory: /home/as/MetONTIIME/fast5_pass_analysis/analysis Flow-cell: FLO-MIN106 Kit: SQK-RAB204 Expected amplicon length [bp]: 1400 Barcodes used in this experiment: BC01, BC02, BC03, BC04, BC05, BC06, BC07, BC08, BC09, BC10, BC11, BC12 Basecalling is going to be performed by : Guppy Basecalling Software, (C) Oxford Nanopore Technologies, Limited. Version 5.0.16+b9fcd7b Basecalling model: fast Demultiplexing is going to be performed by guppy_barcoder after basecalling Taxonomic classifier: Vsearch

Basecalling started at Thu Nov 18 18:03:26 2021 ONT Guppy basecalling software version 5.0.16+b9fcd7b config file: /home/as/ont-guppy-cpu/data/dna_r9.4.1_450bps_fast.cfg model file: /home/as/ont-guppy-cpu/data/template_r9.4.1_450bps_fast.jsn input path: /home/as/MetONTIIME/fast5_pass save path: /home/as/MetONTIIME/fast5_pass_analysis/basecalling chunk size: 2000 chunks per runner: 160 minimum qscore: 8 records per file: 4000 num basecallers: 4 cpu mode: ON threads per caller: 8 Found 10 fast5 files to process. Init time: 82 ms

0% 10 20 30 40 50 60 70 80 90 100% |----|----|----|----|----|----|----|----|----|----|

---

Caller time: 60791 ms, Samples called: 12953296, samples/s: 213079 Finishing up any open output files. Basecalling completed successfully. Basecalling finished at Thu Nov 18 18:04:27 2021

Demultiplexing started at Thu Nov 18 18:04:27 2021

ONT Guppy barcoding software version 5.0.16+b9fcd7b input path: /home/as/MetONTIIME/fast5_pass_analysis/basecalling save path: /home/as/MetONTIIME/fast5_pass_analysis/preprocessing arrangement files: barcode_arrs_rab.cfg lamp arr. files: barcode_arrs_ncov8.cfg barcode_arrs_ncov96.cfg barcode_arrs_multivirus1.cfg barcode_arrs_multivirus8.cfg min. score front: 60 min. score rear: 60

Found 2 input files.

0% 10 20 30 40 50 60 70 80 90 100% |----|----|----|----|----|----|----|----|----|----|

---

Done in 250 ms. Demultiplexing finished at Thu Nov 18 18:04:27 2021

Number of basecalled reads: 1035

Creating folder: /home/as/MetONTIIME/fast5_pass_analysis/qc, which is going to include stats about the sequencing run

Now performing quality control with PycoQC

sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/pycoQC: not found sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found Mean read length (stdev) for sample BC01: NaN (NA) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC01 sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/NanoFilt: not found sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found WARNING: skipping sample BC01, since no reads survived the length or quality filtering!

sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found Mean read length (stdev) for sample BC02: NaN (NA) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC02 sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/NanoFilt: not found sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found WARNING: skipping sample BC02, since no reads survived the length or quality filtering!

sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found Mean read length (stdev) for sample BC03: NaN (NA) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC03 sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/NanoFilt: not found sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found WARNING: skipping sample BC03, since no reads survived the length or quality filtering!

sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found Mean read length (stdev) for sample BC04: NaN (NA) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC04 sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/NanoFilt: not found sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found WARNING: skipping sample BC04, since no reads survived the length or quality filtering!

sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found Mean read length (stdev) for sample BC07: NaN (NA) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC07 sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/NanoFilt: not found sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found WARNING: skipping sample BC07, since no reads survived the length or quality filtering!

sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found Mean read length (stdev) for sample BC08: NaN (NA) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC08 sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/NanoFilt: not found sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found WARNING: skipping sample BC08, since no reads survived the length or quality filtering!

sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found Mean read length (stdev) for sample BC09: NaN (NA) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC09 sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/NanoFilt: not found sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found WARNING: skipping sample BC09, since no reads survived the length or quality filtering!

sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found Mean read length (stdev) for sample BC10: NaN (NA) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC10 sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/NanoFilt: not found sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found WARNING: skipping sample BC10, since no reads survived the length or quality filtering!

sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found Mean read length (stdev) for sample BC11: NaN (NA) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC11 sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/NanoFilt: not found sh: 1: /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk: not found WARNING: skipping sample BC11, since no reads survived the length or quality filtering!

There were 28 warnings (use warnings() to see them) Error in h(simpleError(msg, call)) : error in evaluating the argument 'x' in selecting a method for function 'grep': cannot open the connection Calls: grep -> readLines -> file -> .handleSimpleError -> h In addition: Warning messages: 1: In file(con, "r") : 'raw = FALSE' but '/home/as/MetONTIIME/fast5_pass_analysis/analysis/' is not a regular file 2: In file(con, "r") : cannot open file '/home/as/MetONTIIME/fast5_pass_analysis/analysis/': it is a directory Execution halted

i don't know.... grazie Alessio

[MaestSi]

I think you may have modified these two lines by mistake in Launch_MinION_mobile_lab.sh:

RAW_READS_DIR=fast5_pass
RAW_READS_DIR_FULL=$(./fast5_pass $RAW_READS_DIR)

since the original lines should be:

RAW_READS_DIR=$1
RAW_READS_DIR_FULL=$(realpath $RAW_READS_DIR)

However this should not be the issue that is causing the pipeline to crash. Please type: ls /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk and tell me if it finds the executable. I don't think it is able to find it (and all other executables needed) because you may have an anaconda3 installation conflicting with your miniconda3 installation. You probably have correctly set the variable: MINICONDA_DIR <- "/path/to/miniconda3" in the config_MinION_mobile_lab.R file, but command: which conda may be returning the path to conda in anaconda3 instead of miniconda3. If this is the case, you may have actually installed the MetONTIIME_env environment in anaconda3 instead of miniconda3. Could you please check? If this is the case, everything could be solved easily by adding a line in the .bashrc file to put the path to miniconda3 before the path to anaconda3 in PATH environmental variable, and then repeating the installation. Simone

[asogg]

Thanks Simone , i'll check all soon. Grazie Alessio

[asogg]

exactly as you supposed for the first part

as@as-HP-Pavilion-dv6-Notebook-PC:~$ ls /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk ls: cannot access '/home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk': No such file or directory but with the command which conda it seems that the path is in miniconda

as@as-HP-Pavilion-dv6-Notebook-PC:~$ which conda /home/as/miniconda3/bin/conda

[MaestSi]

Then, just try rerunning: ./install.sh and accepting re-building the environment. In alternative, activate the environment and use the commands you find in the install.sh script to install each dependency, and check if you get any errors. Let me know how it goes! Simone

[asogg]

Hi Simone , i reinstalled with the install.sh arning: 'bioconda' already in 'channels' list, moving to the top Warning: 'conda-forge' already in 'channels' list, moving to the top Warning: 'r' already in 'channels' list, moving to the top Warning: 'anaconda' already in 'channels' list, moving to the top --2021-11-19 09:02:39-- https://data.qiime2.org/distro/core/qiime2-2021.8-py38-linux-conda.yml Resolving data.qiime2.org (data.qiime2.org)... 54.200.1.12 Connecting to data.qiime2.org (data.qiime2.org)|54.200.1.12|:443... connected. HTTP request sent, awaiting response... 302 FOUND Location: https://raw.githubusercontent.com/qiime2/environment-files/master/2021.8/release/qiime2-2021.8-py38-linux-conda.yml [following] --2021-11-19 09:02:40-- https://raw.githubusercontent.com/qiime2/environment-files/master/2021.8/release/qiime2-2021.8-py38-linux-conda.yml Resolving raw.githubusercontent.com (raw.githubusercontent.com)... 185.199.110.133, 185.199.109.133, 185.199.108.133, ... Connecting to raw.githubusercontent.com (raw.githubusercontent.com)|185.199.110.133|:443... connected. HTTP request sent, awaiting response... 200 OK Length: 9224 (9.0K) [text/plain] Saving to: ‘qiime2-2021.8-py38-linux-conda.yml.1’

qiime2-2021.8-py38- 100%[===================>] 9.01K --.-KB/s in 0.005s

2021-11-19 09:02:40 (1.78 MB/s) - ‘qiime2-2021.8-py38-linux-conda.yml.1’ saved [9224/9224]

CondaValueError: prefix already exists: /home/as/miniconda3/envs/MetONTIIME_env

Collecting package metadata (current_repodata.json): done Solving environment: done

Package Plan

environment location: /home/as/miniconda3/envs/MetONTIIME_env

added / updated specs:

• nanofilt
• seqtk

The following packages will be downloaded:

package                    |            build
---------------------------|-----------------
biopython-1.78             |   py38h7b6447c_0         2.6 MB  anaconda
certifi-2020.6.20          |           py38_0         160 KB  anaconda
pysam-0.16.0.1             |   py38hf7546f9_3         2.7 MB  bioconda
wrapt-1.12.1               |   py38h7b6447c_1          50 KB  anaconda
------------------------------------------------------------
                                       Total:         5.5 MB

The following NEW packages will be INSTALLED:

biopython anaconda/linux-64::biopython-1.78-py38h7b6447c_0 deprecated anaconda/noarch::deprecated-1.2.10-py_0 nanofilt bioconda/noarch::nanofilt-2.8.0-py_0 nanoget bioconda/noarch::nanoget-1.16.1-py_0 nanomath bioconda/noarch::nanomath-1.2.1-pyhdfd78af_0 pysam bioconda/linux-64::pysam-0.16.0.1-py38hf7546f9_3 seqtk bioconda/linux-64::seqtk-1.3-h5bf99c6_3 wrapt anaconda/linux-64::wrapt-1.12.1-py38h7b6447c_1

The following packages will be SUPERSEDED by a higher-priority channel:

ca-certificates conda-forge::ca-certificates-2021.5.3~ --> anaconda::ca-certificates-2020.10.14-0 certifi conda-forge::certifi-2021.5.30-py38h5~ --> anaconda::certifi-2020.6.20-py38_0

Proceed ([y]/n)? y

Downloading and Extracting Packages wrapt-1.12.1 | 50 KB | ##################################### | 100% biopython-1.78 | 2.6 MB | ##################################### | 100% certifi-2020.6.20 | 160 KB | ##################################### | 100% pysam-0.16.0.1 | 2.7 MB | ##################################### | 100% Preparing transaction: done Verifying transaction: done Executing transaction: done Collecting pycoQC Downloading pycoQC-2.5.2.tar.gz (35 kB) Requirement already satisfied: numpy>=1.19 in /home/as/miniconda3/envs/MetONTIIME_env/lib/python3.8/site-packages (from pycoQC) (1.21.2) Requirement already satisfied: scipy>=1.5 in /home/as/miniconda3/envs/MetONTIIME_env/lib/python3.8/site-packages (from pycoQC) (1.7.1) Requirement already satisfied: pandas>=1.1 in /home/as/miniconda3/envs/MetONTIIME_env/lib/python3.8/site-packages (from pycoQC) (1.2.5) Collecting plotly==4.1.0 Downloading plotly-4.1.0-py2.py3-none-any.whl (7.1 MB) |████████████████████████████████| 7.1 MB 721 kB/s Requirement already satisfied: jinja2>=2.10 in /home/as/miniconda3/envs/MetONTIIME_env/lib/python3.8/site-packages (from pycoQC) (3.0.1) Requirement already satisfied: h5py>=3.1 in /home/as/miniconda3/envs/MetONTIIME_env/lib/python3.8/site-packages (from pycoQC) (3.3.0) Collecting tqdm>=4.54 Downloading tqdm-4.62.3-py2.py3-none-any.whl (76 kB) |████████████████████████████████| 76 kB 176 kB/s Requirement already satisfied: pysam>=0.16 in /home/as/miniconda3/envs/MetONTIIME_env/lib/python3.8/site-packages (from pycoQC) (0.16.0.1) Collecting retrying>=1.3.3 Downloading retrying-1.3.3.tar.gz (10 kB) Requirement already satisfied: six in /home/as/miniconda3/envs/MetONTIIME_env/lib/python3.8/site-packages (from plotly==4.1.0->pycoQC) (1.16.0) Requirement already satisfied: MarkupSafe>=2.0 in /home/as/miniconda3/envs/MetONTIIME_env/lib/python3.8/site-packages (from jinja2>=2.10->pycoQC) (2.0.1) Requirement already satisfied: python-dateutil>=2.7.3 in /home/as/miniconda3/envs/MetONTIIME_env/lib/python3.8/site-packages (from pandas>=1.1->pycoQC) (2.8.2) Requirement already satisfied: pytz>=2017.3 in /home/as/miniconda3/envs/MetONTIIME_env/lib/python3.8/site-packages (from pandas>=1.1->pycoQC) (2021.1) Building wheels for collected packages: pycoQC, retrying Building wheel for pycoQC (setup.py) ... done Created wheel for pycoQC: filename=pycoQC-2.5.2-py3-none-any.whl size=39551 sha256=26432574deb5b56f54ab903c83df51b478ab66967d025006151f27e68a972a35 Stored in directory: /home/as/.cache/pip/wheels/57/44/55/ae7a414ae2bcc47d07cbc4a292b3e1c6ba2fcaa7fbb39b9154 Building wheel for retrying (setup.py) ... done Created wheel for retrying: filename=retrying-1.3.3-py3-none-any.whl size=11448 sha256=e30537d968bab40f4ddd33f645cdcada13236cf96c1f17260417707e64bca158 Stored in directory: /home/as/.cache/pip/wheels/c4/a7/48/0a434133f6d56e878ca511c0e6c38326907c0792f67b476e56 Successfully built pycoQC retrying Installing collected packages: retrying, tqdm, plotly, pycoQC Successfully installed plotly-4.1.0 pycoQC-2.5.2 retrying-1.3.3 tqdm-4.62.3

Modify variables PIPELINE_DIR and MINICONDA_DIR in config_MinION_mobile_lab.R PIPELINE_DIR <- "/home/as/MetONTIIME" MINICONDA_DIR <- "/home/as/miniconda3

next step run the pipeline with Launch_MinION_mobile_lab.sh correct?

[MaestSi]

Correct! First check with the 'ls' command that the required executables are there! Simone

[asogg]

seems there are as@as-HP-Pavilion-dv6-Notebook-PC:~/MetONTIIME$ ls /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk /home/as/miniconda3/envs/MetONTIIME_env/bin/seqtk as@as-HP-Pavilion-dv6-Notebook-PC:~/MetONTIIME$ ls /home/as/miniconda3/envs/MetONTIIME_env/bin/pycoQC /home/as/miniconda3/envs/MetONTIIME_env/bin/pycoQC as@as-HP-Pavilion-dv6-Notebook-PC:~/MetONTIIME$ ls /home/as/miniconda3/envs/MetONTIIME_env/bin/nanofilt ls: cannot access '/home/as/miniconda3/envs/MetONTIIME_env/bin/nanofilt': No such file or directory as@as-HP-Pavilion-dv6-Notebook-PC:~/MetONTIIME$ ls /home/as/miniconda3/envs/MetONTIIME_env/bin/NanoFilt /home/as/miniconda3/envs/MetONTIIME_env/bin/NanoFilt as@as-HP-Pavilion-dv6-Notebook-PC:~/MetONTIIME$

[asogg]

i started the pipeline with the launch

!/bin/bash

#

Copyright 2021 Simone Maestri. All rights reserved.

Simone Maestri simone.maestri@univr.it

#

This program is free software: you can redistribute it and/or modify

it under the terms of the GNU General Public License as published by

the Free Software Foundation, either version 3 of the License, or

(at your option) any later version.

#

This program is distributed in the hope that it will be useful,

but WITHOUT ANY WARRANTY; without even the implied warranty of

MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the

GNU General Public License for more details.

#

You should have received a copy of the GNU General Public License

along with this program. If not, see http://www.gnu.org/licenses/.

#

RAW_READS_DIR=$1 RAW_READS_DIR_FULL=$(realpath $RAW_READS_DIR) source activate MetONTIIME_env PIPELINE_DIR=$( /home/as/MetONTIIME $( dirname "${BASH_SOURCE[0]}" )) nohup Rscript /home/as/MetONTIIME/MinION_mobile_lab.R /home/as/MetONTIIME/config_MinION_mobile_lab.R /home/as/MetONTIIME/fast5_pass

s@as-HP-Pavilion-dv6-Notebook-PC:~/MetONTIIME$ ./Launch_MinION_mobile_lab.sh realpath: missing operand Try 'realpath --help' for more information. ./Launch_MinION_mobile_lab.sh: line 24: /home/as/MetONTIIME: Is a directory nohup: ignoring input and appending output to 'nohup.out'

Welcome to MinION meta-barcoding mobile laboratory!

Raw reads directory: /home/as/MetONTIIME/fast5_pass Basecalled reads directory: /home/as/MetONTIIME/fast5_pass_analysis/basecalling Preprocessing directory: /home/as/MetONTIIME/fast5_pass_analysis/preprocessing Analysis and results directory: /home/as/MetONTIIME/fast5_pass_analysis/analysis Flow-cell: FLO-MIN106 Kit: SQK-RAB204 Expected amplicon length [bp]: 1400 Barcodes used in this experiment: BC01, BC02, BC03, BC04, BC05, BC06, BC07, BC08, BC09, BC10, BC11, BC12 Basecalling is going to be performed by : Guppy Basecalling Software, (C) Oxford Nanopore Technologies, Limited. Version 5.0.16+b9fcd7b Basecalling model: fast Demultiplexing is going to be performed by guppy_barcoder after basecalling Taxonomic classifier: Vsearch

Basecalling started at Fri Nov 19 11:15:26 2021 ONT Guppy basecalling software version 5.0.16+b9fcd7b config file: /home/as/ont-guppy-cpu/data/dna_r9.4.1_450bps_fast.cfg model file: /home/as/ont-guppy-cpu/data/template_r9.4.1_450bps_fast.jsn input path: /home/as/MetONTIIME/fast5_pass save path: /home/as/MetONTIIME/fast5_pass_analysis/basecalling chunk size: 2000 chunks per runner: 160 minimum qscore: 8 records per file: 4000 num basecallers: 4 cpu mode: ON threads per caller: 8 Found 10 fast5 files to process. Init time: 67 ms

0% 10 20 30 40 50 60 70 80 90 100% |----|----|----|----|----|----|----|----|----|----|

---

Caller time: 58835 ms, Samples called: 12953296, samples/s: 220163 Finishing up any open output files. Basecalling completed successfully. Basecalling finished at Fri Nov 19 11:16:25 2021

Demultiplexing started at Fri Nov 19 11:16:25 2021

ONT Guppy barcoding software version 5.0.16+b9fcd7b input path: /home/as/MetONTIIME/fast5_pass_analysis/basecalling save path: /home/as/MetONTIIME/fast5_pass_analysis/preprocessing arrangement files: barcode_arrs_rab.cfg lamp arr. files: barcode_arrs_ncov8.cfg barcode_arrs_ncov96.cfg barcode_arrs_multivirus1.cfg barcode_arrs_multivirus8.cfg min. score front: 60 min. score rear: 60

Found 2 input files.

0% 10 20 30 40 50 60 70 80 90 100% |----|----|----|----|----|----|----|----|----|----|

---

Done in 243 ms. Demultiplexing finished at Fri Nov 19 11:16:25 2021

Number of basecalled reads: 1035

Creating folder: /home/as/MetONTIIME/fast5_pass_analysis/qc, which is going to include stats about the sequencing run

Now performing quality control with PycoQC

Checking arguments values Check input data files Parse data files Merge data Cleaning data Discarding lines containing NA values 0 reads discarded Filtering out zero length reads 0 reads discarded Sorting run IDs by decreasing throughput Run-id order ['022704de63ea194d22d08bd1f600dde4442d8a82'] Reordering runids Processing reads with Run_ID 022704de63ea194d22d08bd1f600dde4442d8a82 / time offset: 0 Cleaning up low frequency barcodes 0 reads with low frequency barcode unset Cast value to appropriate type Reindexing dataframe by read_ids 1,033 Final valid reads Loading plotting interface Found 1,033 total reads Found 1,032 pass reads (qual >= 7.0 and length >= 0) Generating HTML report Parsing html config file Running method run_summary Computing plot Running method basecall_summary Computing plot Running method alignment_summary No Alignment information available Running method read_len_1D Computing plot Running method align_len_1D No Alignment information available Running method read_qual_1D Computing plot Running method identity_freq_1D No identity frequency information available Running method read_len_read_qual_2D Computing plot Running method read_len_align_len_2D No Alignment information available Running method align_len_identity_freq_2D No identity frequency information available Running method read_qual_identity_freq_2D No identity frequency information available Running method output_over_time Computing plot Running method read_len_over_time Computing plot Running method read_qual_over_time Computing plot Running method align_len_over_time No Alignment information available Running method identity_freq_over_time No identity frequency information available Running method barcode_counts Computing plot Running method channels_activity Computing plot Running method alignment_reads_status No Alignment information available Running method alignment_rate No identity frequency information available Running method alignment_coverage No Alignment information available Loading HTML template Rendering plots in d3js Writing to HTML file Mean read length (stdev) for sample BC01: 1163 (476) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC01 Mean read length for sample BC01 after filtering: 1385 (45) Mean read length (stdev) for sample BC02: 522 (635) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC02 Mean read length for sample BC02 after filtering: 1407 (51) Mean read length (stdev) for sample BC03: 671 (645) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC03 Mean read length for sample BC03 after filtering: 1395 (38) Mean read length (stdev) for sample BC04: 1109 (564) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC04 Mean read length for sample BC04 after filtering: 1407 (39) Mean read length (stdev) for sample BC07: 1363 (58) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC07 Mean read length for sample BC07 after filtering: 1363 (58) Mean read length (stdev) for sample BC08: 1420 (53) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC08 Mean read length for sample BC08 after filtering: 1420 (53) Mean read length (stdev) for sample BC09: 1416 (NA) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC09 Mean read length for sample BC09 after filtering: 1416 (NA) Mean read length (stdev) for sample BC10: 1389 (31) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC10 Mean read length for sample BC10 after filtering: 1389 (31) Mean read length (stdev) for sample BC11: 1397 (NA) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC11 Mean read length for sample BC11 after filtering: 1397 (NA) Number of reads assigned to BC01: 440 Number of reads assigned to BC02: 14 Number of reads assigned to BC03: 32 Number of reads assigned to BC04: 103 Number of reads assigned to BC07: 6 Number of reads assigned to BC08: 4 Number of reads assigned to BC09: 1 Number of reads assigned to BC10: 8 Number of reads assigned to BC11: 1

Compressing BC01.fastq file Compressing BC02.fastq file Compressing BC03.fastq file Compressing BC04.fastq file Compressing BC07.fastq file Compressing BC08.fastq file Compressing BC09.fastq file Compressing BC10.fastq file Compressing BC11.fastq file

Running the MetONTIIME pipeline

sh: 1: /home/as/MetONTIIME/MetONTIIME.sh: Permission denied Workflow ended at Fri Nov 19 11:16:40 2021! Look at the results in directory /home/as/MetONTIIME/fast5_pass_analysis/analysis

[asogg]

i changed the file permission of metontiime.sh with chmod +x and seems it is working! Welcome to MinION meta-barcoding mobile laboratory!

Raw reads directory: /home/as/MetONTIIME/fast5_pass Basecalled reads directory: /home/as/MetONTIIME/fast5_pass_analysis/basecalling Preprocessing directory: /home/as/MetONTIIME/fast5_pass_analysis/preprocessing Analysis and results directory: /home/as/MetONTIIME/fast5_pass_analysis/analysis Flow-cell: FLO-MIN106 Kit: SQK-RAB204 Expected amplicon length [bp]: 1400 Barcodes used in this experiment: BC01, BC02, BC03, BC04, BC05, BC06, BC07, BC08, BC09, BC10, BC11, BC12 Basecalling is going to be performed by : Guppy Basecalling Software, (C) Oxford Nanopore Technologies, Limited. Version 5.0.16+b9fcd7b Basecalling model: fast Demultiplexing is going to be performed by guppy_barcoder after basecalling Taxonomic classifier: Vsearch

Basecalling started at Fri Nov 19 11:23:16 2021 ONT Guppy basecalling software version 5.0.16+b9fcd7b config file: /home/as/ont-guppy-cpu/data/dna_r9.4.1_450bps_fast.cfg model file: /home/as/ont-guppy-cpu/data/template_r9.4.1_450bps_fast.jsn input path: /home/as/MetONTIIME/fast5_pass save path: /home/as/MetONTIIME/fast5_pass_analysis/basecalling chunk size: 2000 chunks per runner: 160 minimum qscore: 8 records per file: 4000 num basecallers: 4 cpu mode: ON threads per caller: 8 Found 10 fast5 files to process. Init time: 62 ms

0% 10 20 30 40 50 60 70 80 90 100% |----|----|----|----|----|----|----|----|----|----|

---

Caller time: 60069 ms, Samples called: 12953296, samples/s: 215640 Finishing up any open output files. Basecalling completed successfully. Basecalling finished at Fri Nov 19 11:24:16 2021

Demultiplexing started at Fri Nov 19 11:24:16 2021

ONT Guppy barcoding software version 5.0.16+b9fcd7b input path: /home/as/MetONTIIME/fast5_pass_analysis/basecalling save path: /home/as/MetONTIIME/fast5_pass_analysis/preprocessing arrangement files: barcode_arrs_rab.cfg lamp arr. files: barcode_arrs_ncov8.cfg barcode_arrs_ncov96.cfg barcode_arrs_multivirus1.cfg barcode_arrs_multivirus8.cfg min. score front: 60 min. score rear: 60

Found 2 input files.

0% 10 20 30 40 50 60 70 80 90 100% |----|----|----|----|----|----|----|----|----|----|

---

Done in 264 ms. Demultiplexing finished at Fri Nov 19 11:24:17 2021

Number of basecalled reads: 1035

Creating folder: /home/as/MetONTIIME/fast5_pass_analysis/qc, which is going to include stats about the sequencing run

Now performing quality control with PycoQC

Checking arguments values Check input data files Parse data files Merge data Cleaning data Discarding lines containing NA values 0 reads discarded Filtering out zero length reads 0 reads discarded Sorting run IDs by decreasing throughput Run-id order ['022704de63ea194d22d08bd1f600dde4442d8a82'] Reordering runids Processing reads with Run_ID 022704de63ea194d22d08bd1f600dde4442d8a82 / time offset: 0 Cleaning up low frequency barcodes 0 reads with low frequency barcode unset Cast value to appropriate type Reindexing dataframe by read_ids 1,033 Final valid reads Loading plotting interface Found 1,033 total reads Found 1,032 pass reads (qual >= 7.0 and length >= 0) Generating HTML report Parsing html config file Running method run_summary Computing plot Running method basecall_summary Computing plot Running method alignment_summary No Alignment information available Running method read_len_1D Computing plot Running method align_len_1D No Alignment information available Running method read_qual_1D Computing plot Running method identity_freq_1D No identity frequency information available Running method read_len_read_qual_2D Computing plot Running method read_len_align_len_2D No Alignment information available Running method align_len_identity_freq_2D No identity frequency information available Running method read_qual_identity_freq_2D No identity frequency information available Running method output_over_time Computing plot Running method read_len_over_time Computing plot Running method read_qual_over_time Computing plot Running method align_len_over_time No Alignment information available Running method identity_freq_over_time No identity frequency information available Running method barcode_counts Computing plot Running method channels_activity Computing plot Running method alignment_reads_status No Alignment information available Running method alignment_rate No identity frequency information available Running method alignment_coverage No Alignment information available Loading HTML template Rendering plots in d3js Writing to HTML file Mean read length (stdev) for sample BC01: 1163 (476) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC01 Mean read length for sample BC01 after filtering: 1385 (45) Mean read length (stdev) for sample BC02: 522 (635) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC02 Mean read length for sample BC02 after filtering: 1407 (51) Mean read length (stdev) for sample BC03: 671 (645) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC03 Mean read length for sample BC03 after filtering: 1395 (38) Mean read length (stdev) for sample BC04: 1109 (564) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC04 Mean read length for sample BC04 after filtering: 1407 (39) Mean read length (stdev) for sample BC07: 1363 (58) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC07 Mean read length for sample BC07 after filtering: 1363 (58) Mean read length (stdev) for sample BC08: 1420 (53) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC08 Mean read length for sample BC08 after filtering: 1420 (53) Mean read length (stdev) for sample BC09: 1416 (NA) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC09 Mean read length for sample BC09 after filtering: 1416 (NA) Mean read length (stdev) for sample BC10: 1389 (31) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC10 Mean read length for sample BC10 after filtering: 1389 (31) Mean read length (stdev) for sample BC11: 1397 (NA) Now filtering out reads with quality lower than 7, shorter than 1250 and longer than 1550 bp for sample BC11 Mean read length for sample BC11 after filtering: 1397 (NA) Number of reads assigned to BC01: 440 Number of reads assigned to BC02: 14 Number of reads assigned to BC03: 32 Number of reads assigned to BC04: 103 Number of reads assigned to BC07: 6 Number of reads assigned to BC08: 4 Number of reads assigned to BC09: 1 Number of reads assigned to BC10: 8 Number of reads assigned to BC11: 1

Compressing BC01.fastq file Compressing BC02.fastq file Compressing BC03.fastq file Compressing BC04.fastq file Compressing BC07.fastq file Compressing BC08.fastq file Compressing BC09.fastq file Compressing BC10.fastq file Compressing BC11.fastq file

Running the MetONTIIME pipeline

Imported /home/as/MetONTIIME/fast5_pass_analysis/analysis/manifest.txt as SingleEndFastqManifestPhred33V2 to sequences.qza Saved FeatureTable[Frequency] to: table_tmp.qza Saved FeatureData[Sequence] to: rep-seqs_tmp.qza Saved FeatureTable[Frequency] to: table.qza Saved FeatureData[Sequence] to: rep-seqs.qza Saved Visualization to: demux_summary.qzv Saved Visualization to: table.qzv Saved Visualization to: rep-seqs.qzv Saved FeatureData[Taxonomy] to: taxonomy.qza Saved Visualization to: taxonomy.qzv Saved Visualization to: taxa-bar-plots.qzv Saved FeatureTable[Frequency] to: table-no-Unassigned.qza Saved Visualization to: taxa-bar-plots-no-Unassigned.qzv Saved FeatureTable[Frequency] to: table_collapsed_absfreq_level1.qza Saved Visualization to: table_collapsed_absfreq_level1.qzv Exported table_collapsed_absfreq_level1.qza as BIOMV210DirFmt to directory . Saved FeatureTable[RelativeFrequency] to: table_collapsed_relfreq_level1.qza Saved Visualization to: table_collapsed_relfreq_level1.qzv Exported table_collapsed_relfreq_level1.qza as BIOMV210DirFmt to directory . Saved FeatureTable[Frequency] to: table_collapsed_absfreq_level2.qza Saved Visualization to: table_collapsed_absfreq_level2.qzv Exported table_collapsed_absfreq_level2.qza as BIOMV210DirFmt to directory . Saved FeatureTable[RelativeFrequency] to: table_collapsed_relfreq_level2.qza Saved Visualization to: table_collapsed_relfreq_level2.qzv Exported table_collapsed_relfreq_level2.qza as BIOMV210DirFmt to directory . Saved FeatureTable[Frequency] to: table_collapsed_absfreq_level3.qza Saved Visualization to: table_collapsed_absfreq_level3.qzv Exported table_collapsed_absfreq_level3.qza as BIOMV210DirFmt to directory . Saved FeatureTable[RelativeFrequency] to: table_collapsed_relfreq_level3.qza Saved Visualization to: table_collapsed_relfreq_level3.qzv Exported table_collapsed_relfreq_level3.qza as BIOMV210DirFmt to directory . Saved FeatureTable[Frequency] to: table_collapsed_absfreq_level4.qza Saved Visualization to: table_collapsed_absfreq_level4.qzv Exported table_collapsed_absfreq_level4.qza as BIOMV210DirFmt to directory . Saved FeatureTable[RelativeFrequency] to: table_collapsed_relfreq_level4.qza Saved Visualization to: table_collapsed_relfreq_level4.qzv Exported table_collapsed_relfreq_level4.qza as BIOMV210DirFmt to directory . Saved FeatureTable[Frequency] to: table_collapsed_absfreq_level5.qza Saved Visualization to: table_collapsed_absfreq_level5.qzv Exported table_collapsed_absfreq_level5.qza as BIOMV210DirFmt to directory . Saved FeatureTable[RelativeFrequency] to: table_collapsed_relfreq_level5.qza Saved Visualization to: table_collapsed_relfreq_level5.qzv Exported table_collapsed_relfreq_level5.qza as BIOMV210DirFmt to directory . Saved FeatureTable[Frequency] to: table_collapsed_absfreq_level6.qza Saved Visualization to: table_collapsed_absfreq_level6.qzv Exported table_collapsed_absfreq_level6.qza as BIOMV210DirFmt to directory . Saved FeatureTable[RelativeFrequency] to: table_collapsed_relfreq_level6.qza Saved Visualization to: table_collapsed_relfreq_level6.qzv Exported table_collapsed_relfreq_level6.qza as BIOMV210DirFmt to directory . Saved FeatureTable[Frequency] to: table_collapsed_absfreq_level7.qza Saved Visualization to: table_collapsed_absfreq_level7.qzv Exported table_collapsed_absfreq_level7.qza as BIOMV210DirFmt to directory . Saved FeatureTable[RelativeFrequency] to: table_collapsed_relfreq_level7.qza Saved Visualization to: table_collapsed_relfreq_level7.qzv Exported table_collapsed_relfreq_level7.qza as BIOMV210DirFmt to directory . Workflow ended at Fri Nov 19 11:30:48 2021! Look at the results in directory /home/as/MetONTIIME/fast5_pass_analysis/analysis

what do you think grazie Alessio

[MaestSi]

Yes, I think it is working as expected! Try uploading to qiime2 viewer the file taxa-bar-plots.qzv and check if you can see the bars corresponding to each sample. If yes, then I think we can close the issue. Ciao, Simone

[asogg]

yes , it works ! i will move to the other functions (eg. evaluate diversity). we keep in touch thank you so much grazie mille Alessio

[MaestSi]

You're welcome! Simone