Ion Torrent data - trim and quality filter

#1

Hello QIIME2 users,

I beginner working on bioinformatics. I am planning to use QIIME2 for 16S rRNA sequencing analysis (bacterial composition, bacterial diversity, etc.).

I have 15 .fastq sequences files, were obtained from Ion 16S Metagenoimics kit with Ion Torrent machine as following:

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Also,
I imported .fastq files to QIIME2 followed the Ion Torrent Importing data by using “Fastq manifest” formats and now I got .qza file.

I have used this command line for import:
qiime tools import
–type SampleData[JoinedSequencesWithQuality]
–input-path manifest_IonXpressBV_ED.txt
–output-path single-end-demux-BV-JoinedSeq.qza
–input-format SingleEndFastqManifestPhred33

After importing data, I checked peek:
qiime tools peek single-end-demux.qza
UUID: f228347a-1913-48c7-b037-a519869e1001
Type: SampleData[JoinedSequencesWithQuality]
Data format: SingleLanePerSampleSingleEndFastqDirFmt

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Then I will process to check sequence quality control by using quality-filter q-score-joined but I got an error message. What wrong in this step? and would like some advice?

I have used this for check quality-filter:
qiime quality-filter q-score-joined
–i-demux single-end-demux-BV-JoinedSeq.qza
–o-filtered-sequences BV-demux-joined-filtered.qza
–o-filter-stats BV-demux-joined-filter-stats.qza

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In addition, I am trying to denoise with q2-deblur, qiime deblur denoise-16S, followed Ion Torrent data denoise with dada2 but I got error from deblur.

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How to proceed further in this case?
Please give more information and suggestion.

Regards,
Auttawit

#2

Can you run the following to check your data?

qiime tools validate single-end-demux-BV-JoinedSeq.qza

Thanks! :qiime2: :t_rex:

#3

Hi @thermokarst,

I checked following your suggestion but it showed some error.

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Do you have any suggestion?

Thank you very much for your help.
Regards,
Auttawit

#4
#5

I suspect this might be a locale issue - we can check that by running the following, and pasting the results here:

env

Thanks! :qiime2:

#6
#7

Hi @thermokarst,

I followed your suggestion, the results as below

(qiime2-2019.7) [email protected]:/media/sf_sf_Desktop/Raw Data$ env
CLUTTER_IM_MODULE=xim
CONDA_SHLVL=1
LC_ALL=C.UTF-8
LS_COLORS=rs=0:di=01;34:ln=01;36:mh=00:pi=40;33:so=01;35:do=01;35:bd=40;33;01:cd=40;33;01:or=40;31;01:mi=00:su=37;41:sg=30;43:ca=30;41:tw=30;42:ow=34;42:st=37;44:ex=01;32:.tar=01;31:.tgz=01;31:.arc=01;31:.arj=01;31:.taz=01;31:.lha=01;31:.lz4=01;31:.lzh=01;31:.lzma=01;31:.tlz=01;31:.txz=01;31:.tzo=01;31:.t7z=01;31:.zip=01;31:.z=01;31:.Z=01;31:.dz=01;31:.gz=01;31:.lrz=01;31:.lz=01;31:.lzo=01;31:.xz=01;31:.zst=01;31:.tzst=01;31:.bz2=01;31:.bz=01;31:.tbz=01;31:.tbz2=01;31:.tz=01;31:.deb=01;31:.rpm=01;31:.jar=01;31:.war=01;31:.ear=01;31:.sar=01;31:.rar=01;31:.alz=01;31:.ace=01;31:.zoo=01;31:.cpio=01;31:.7z=01;31:.rz=01;31:.cab=01;31:.wim=01;31:.swm=01;31:.dwm=01;31:.esd=01;31:.jpg=01;35:.jpeg=01;35:.mjpg=01;35:.mjpeg=01;35:.gif=01;35:.bmp=01;35:.pbm=01;35:.pgm=01;35:.ppm=01;35:.tga=01;35:.xbm=01;35:.xpm=01;35:.tif=01;35:.tiff=01;35:.png=01;35:.svg=01;35:.svgz=01;35:.mng=01;35:.pcx=01;35:.mov=01;35:.mpg=01;35:.mpeg=01;35:.m2v=01;35:.mkv=01;35:.webm=01;35:.ogm=01;35:.mp4=01;35:.m4v=01;35:.mp4v=01;35:.vob=01;35:.qt=01;35:.nuv=01;35:.wmv=01;35:.asf=01;35:.rm=01;35:.rmvb=01;35:.flc=01;35:.avi=01;35:.fli=01;35:.flv=01;35:.gl=01;35:.dl=01;35:.xcf=01;35:.xwd=01;35:.yuv=01;35:.cgm=01;35:.emf=01;35:.ogv=01;35:.ogx=01;35:.aac=00;36:.au=00;36:.flac=00;36:.m4a=00;36:.mid=00;36:.midi=00;36:.mka=00;36:.mp3=00;36:.mpc=00;36:.ogg=00;36:.ra=00;36:.wav=00;36:.oga=00;36:.opus=00;36:.spx=00;36:.xspf=00;36:
CONDA_EXE=/home/qiime2/miniconda/bin/conda
LESSCLOSE=/usr/bin/lesspipe %s %s
GPROF=/home/qiime2/miniconda/envs/qiime2-2019.7/bin/x86_64-conda_cos6-linux-gnu-gprof
XDG_MENU_PREFIX=gnome-
LANG=C.UTF-8
MANAGERPID=2147
DISPLAY=:0
GXX=/home/qiime2/miniconda/envs/qiime2-2019.7/bin/x86_64-conda_cos6-linux-gnu-g++
JAVA_LD_LIBRARY_PATH=/home/qiime2/miniconda/envs/qiime2-2019.7/lib/server
INVOCATION_ID=9c5d5976dcc1413185ae99b4481d470f
LD_GOLD=/home/qiime2/miniconda/envs/qiime2-2019.7/bin/x86_64-conda_cos6-linux-gnu-ld.gold
GNOME_SHELL_SESSION_MODE=ubuntu
DEBUG_CPPFLAGS=-D_DEBUG -D_FORTIFY_SOURCE=2 -Og
DEBUG_CFLAGS=-march=nocona -mtune=haswell -ftree-vectorize -fPIC -fstack-protector-all -fno-plt -Og -g -Wall -Wextra -fvar-tracking-assignments -ffunction-sections -pipe
COLORTERM=truecolor
GCC_NM=/home/qiime2/miniconda/envs/qiime2-2019.7/bin/x86_64-conda_cos6-linux-gnu-gcc-nm
USERNAME=qiime2
CONDA_PREFIX=/home/qiime2/miniconda/envs/qiime2-2019.7
JAVA_HOME=/home/qiime2/miniconda/envs/qiime2-2019.7
GCC_RANLIB=/home/qiime2/miniconda/envs/qiime2-2019.7/bin/x86_64-conda_cos6-linux-gnu-gcc-ranlib
CFLAGS=-march=nocona -mtune=haswell -ftree-vectorize -fPIC -fstack-protector-strong -fno-plt -O2 -ffunction-sections -pipe
GCC_AR=/home/qiime2/miniconda/envs/qiime2-2019.7/bin/x86_64-conda_cos6-linux-gnu-gcc-ar
R_LIBS_USER=/home/qiime2/miniconda/envs/qiime2-2019.7/lib/R/library/
XDG_VTNR=2
SSH_AUTH_SOCK=/run/user/1001/keyring/ssh
MANDATORY_PATH=/usr/share/gconf/ubuntu.mandatory.path
_CE_M=
CC=/home/qiime2/miniconda/envs/qiime2-2019.7/bin/x86_64-conda_cos6-linux-gnu-cc
READELF=/home/qiime2/miniconda/envs/qiime2-2019.7/bin/x86_64-conda_cos6-linux-gnu-readelf
XDG_SESSION_ID=2
ARBHOME=/home/qiime2/miniconda/envs/qiime2-2019.7/lib/arb
USER=qiime2
CXXFLAGS=-fvisibility-inlines-hidden -std=c++17 -fmessage-length=0 -march=nocona -mtune=haswell -ftree-vectorize -fPIC -fstack-protector-strong -fno-plt -O2 -ffunction-sections -pipe
ARBHOME_BACKUP=
DESKTOP_SESSION=ubuntu
STRIP=/home/qiime2/miniconda/envs/qiime2-2019.7/bin/x86_64-conda_cos6-linux-gnu-strip
DEBUG_FFLAGS=-fopenmp -march=nocona -mtune=haswell -ftree-vectorize -fPIC -fstack-protector-strong -fno-plt -O2 -ffunction-sections -pipe -fopenmp -march=nocona -mtune=haswell -ftree-vectorize -fPIC -fstack-protector-all -fno-plt -Og -g -Wall -Wextra -fcheck=all -fbacktrace -fimplicit-none -fvar-tracking-assignments -ffunction-sections -pipe
OBJCOPY=/home/qiime2/miniconda/envs/qiime2-2019.7/bin/x86_64-conda_cos6-linux-gnu-objcopy
_CONDA_PYTHON_SYSCONFIGDATA_NAME=_sysconfigdata_x86_64_conda_cos6_linux_gnu
QT4_IM_MODULE=xim
TEXTDOMAINDIR=/usr/share/locale/
PYTHONNOUSERSITE=/home/qiime2/miniconda/envs/qiime2-2019.7/lib/python*/site-packages/
GNOME_TERMINAL_SCREEN=/org/gnome/Terminal/screen/2e5ca721_d48f_4948_a3c4_95c73537d65f
DEFAULTS_PATH=/usr/share/gconf/ubuntu.default.path
PWD=/media/sf_sf_Desktop/Raw Data
HOME=/home/qiime2
ADDR2LINE=/home/qiime2/miniconda/envs/qiime2-2019.7/bin/x86_64-conda_cos6-linux-gnu-addr2line
CONDA_PYTHON_EXE=/home/qiime2/miniconda/bin/python
JOURNAL_STREAM=9:28872
TEXTDOMAIN=im-config
SSH_AGENT_PID=2314
HOST=x86_64-conda_cos6-linux-gnu
QT_ACCESSIBILITY=1
XDG_SESSION_TYPE=x11
F77=/home/qiime2/miniconda/envs/qiime2-2019.7/bin/x86_64-conda_cos6-linux-gnu-gfortran
FORTRANFLAGS=-fopenmp -march=nocona -mtune=haswell -ftree-vectorize -fPIC -fstack-protector-strong -fno-plt -O2 -ffunction-sections -pipe
XDG_DATA_DIRS=/usr/share/ubuntu:/usr/local/share/:/usr/share/:/var/lib/snapd/desktop
RANLIB=/home/qiime2/miniconda/envs/qiime2-2019.7/bin/x86_64-conda_cos6-linux-gnu-ranlib
OBJDUMP=/home/qiime2/miniconda/envs/qiime2-2019.7/bin/x86_64-conda_cos6-linux-gnu-objdump
AS=/home/qiime2/miniconda/envs/qiime2-2019.7/bin/x86_64-conda_cos6-linux-gnu-as
AR=/home/qiime2/miniconda/envs/qiime2-2019.7/bin/x86_64-conda_cos6-linux-gnu-ar
GFORTRAN=/home/qiime2/miniconda/envs/qiime2-2019.7/bin/x86_64-conda_cos6-linux-gnu-gfortran
CONDA_BACKUP_HOST=x86_64-conda_cos6-linux-gnu
_CE_CONDA=
XDG_SESSION_DESKTOP=ubuntu
DBUS_STARTER_ADDRESS=unix:path=/run/user/1001/bus,guid=f543adef1306121403a62e1d5dcc1dcd
DEBUG_FORTRANFLAGS=-fopenmp -march=nocona -mtune=haswell -ftree-vectorize -fPIC -fstack-protector-strong -fno-plt -O2 -ffunction-sections -pipe -fopenmp -march=nocona -mtune=haswell -ftree-vectorize -fPIC -fstack-protector-all -fno-plt -Og -g -Wall -Wextra -fcheck=all -fbacktrace -fimplicit-none -fvar-tracking-assignments -ffunction-sections -pipe
FC=/home/qiime2/miniconda/envs/qiime2-2019.7/bin/x86_64-conda_cos6-linux-gnu-gfortran
NM=/home/qiime2/miniconda/envs/qiime2-2019.7/bin/x86_64-conda_cos6-linux-gnu-nm
CONDA_PROMPT_MODIFIER=(qiime2-2019.7)
GTK_MODULES=gail:atk-bridge
DEBUG_CXXFLAGS=-fvisibility-inlines-hidden -std=c++17 -fmessage-length=0 -march=nocona -mtune=haswell -ftree-vectorize -fPIC -fstack-protector-all -fno-plt -Og -g -Wall -Wextra -fvar-tracking-assignments -ffunction-sections -pipe
CXX=/home/qiime2/miniconda/envs/qiime2-2019.7/bin/x86_64-conda_cos6-linux-gnu-c++
WINDOWPATH=2
TERM=xterm-256color
SHELL=/bin/bash
VTE_VERSION=5202
CXXFILT=/home/qiime2/miniconda/envs/qiime2-2019.7/bin/x86_64-conda_cos6-linux-gnu-c++filt
QT_IM_MODULE=xim
[email protected]=ibus
MPLBACKEND=Agg
IM_CONFIG_PHASE=2
DBUS_STARTER_BUS_TYPE=session
XDG_CURRENT_DESKTOP=ubuntu:GNOME
GPG_AGENT_INFO=/run/user/1001/gnupg/S.gpg-agent:0:1
GNOME_TERMINAL_SERVICE=:1.71
ELFEDIT=/home/qiime2/miniconda/envs/qiime2-2019.7/bin/x86_64-conda_cos6-linux-gnu-elfedit
XDG_SEAT=seat0
SHLVL=1
LANGUAGE=en_US:
FFLAGS=-fopenmp -march=nocona -mtune=haswell -ftree-vectorize -fPIC -fstack-protector-strong -fno-plt -O2 -ffunction-sections -pipe
CPPFLAGS=-DNDEBUG -D_FORTIFY_SOURCE=2 -O2
STRINGS=/home/qiime2/miniconda/envs/qiime2-2019.7/bin/x86_64-conda_cos6-linux-gnu-strings
F90=/home/qiime2/miniconda/envs/qiime2-2019.7/bin/x86_64-conda_cos6-linux-gnu-gfortran
F95=/home/qiime2/miniconda/envs/qiime2-2019.7/bin/x86_64-conda_cos6-linux-gnu-f95
GDMSESSION=ubuntu
GNOME_DESKTOP_SESSION_ID=this-is-deprecated
LOGNAME=qiime2
DBUS_SESSION_BUS_ADDRESS=unix:path=/run/user/1001/bus,guid=f543adef1306121403a62e1d5dcc1dcd
JAVA_HOME_CONDA_BACKUP=
XDG_RUNTIME_DIR=/run/user/1001
XAUTHORITY=/run/user/1001/gdm/Xauthority
RSTUDIO_WHICH_R=/home/qiime2/miniconda/envs/qiime2-2019.7/bin/R
LDFLAGS=-Wl,-O2 -Wl,–sort-common -Wl,–as-needed -Wl,-z,relro -Wl,-z,now -Wl,–disable-new-dtags -Wl,–gc-sections
XDG_CONFIG_DIRS=/etc/xdg/xdg-ubuntu:/etc/xdg
PATH=/home/qiime2/miniconda/envs/qiime2-2019.7/bin:/home/qiime2/miniconda/condabin:/home/qiime2/miniconda/bin:/usr/local/bin:/usr/bin:/bin:/usr/local/games:/usr/games:/snap/bin
GCC=/home/qiime2/miniconda/envs/qiime2-2019.7/bin/x86_64-conda_cos6-linux-gnu-gcc
CONDA_DEFAULT_ENV=qiime2-2019.7
SIZE=/home/qiime2/miniconda/envs/qiime2-2019.7/bin/x86_64-conda_cos6-linux-gnu-size
SESSION_MANAGER=local/qiime2core2019-7:@/tmp/.ICE-unix/2179,unix/qiime2core2019-7:/tmp/.ICE-unix/2179
CPP=/home/qiime2/miniconda/envs/qiime2-2019.7/bin/x86_64-conda_cos6-linux-gnu-cpp
LD=/home/qiime2/miniconda/envs/qiime2-2019.7/bin/x86_64-conda_cos6-linux-gnu-ld
LESSOPEN=| /usr/bin/lesspipe %s
JAVA_LD_LIBRARY_PATH_BACKUP=
GTK_IM_MODULE=ibus
_=/usr/bin/env
(qiime2-2019.7) [email protected]:/media/sf_sf_Desktop/Raw Data$

Thank you very much for your help.
Regards,
Auttawit