Package: novowrap
Quick startDownloadthe package,
unzip, and then double-clicknovowrap.exeornovowrap.ORMake sure you havePython(3.7 or higher) orcondainstalled.Open terminal, run# Install, using pip (recommended)pipinstallnovowrap--user# Or, use condacondainstall-cwpwupingwpnovowrap# Initiliaze with Internet# Windowspython-mnovowrapinit# Linux and MacOSpython3-mnovowrapinit# Run# Windowspython-mnovowrap# Linux and MacOSpython3-mnovowrapTable of ContentsQuick startFeaturePrerequisiteHardwareSoftwareInstallationPortableInstall with pipInstall with condaInitializationUsageCommand lineGraphical user interfaceInputOutputOptionsAssemblyValidateMergePerformanceFeature:heavy_check_mark: Assembly chloroplast genomes from given NGS data, with minimal
parameters to set. Also, it supports batch mode.Automatic generate uniform structure with reference (typically, start fromtrnH-psbA, and, SSC/LSC region have the same direction with reference).:heavy_check_mark: Merge contigs according to overlapping region. May handle
Invert-Repeat fragments.:heavy_check_mark: Validate assembly results by comparing the synteny and sequence
homology with given reference (or taxonomy name).PrerequisiteHardwareThe assembly function will call NOVOPlasty, which requires 2 GB memory for 1
GB uncompressed data.The other functions could run in normal computers and have no extra
requirements for memory, CPU, et al.The software requires Internet for the first run to install the missing
dependencies. Then, it could work if offline, but better with the internet.SoftwareFor the portable version, nothing needs to be installed manually.For installing from pip,Pythonis
required. Notice that the python version should be3.7or higher.:white_check_mark: All third-party dependencies will be automatically
installed with the Internet, includingbiopython,matplotlib,coloredlogs,graphviz(python packages), andperl(for Windows only),NOVOPlasty,BLAST.InstallationPortableDownload from thelink,
unpack and run with Internet for the first time.Install with pipInstallPython.3.7 or neweris
required.Open the command line, runpipinstallnovowrap--userInstall with condaAfter installedconda, runconda install -c wpwupingwp novowrapInitializationDuring the first running,novowrapwill check and initialize the running environment.
Missing dependencies will be automatically installed.
This step requires the Internet connection.# Windowspython-mnovowrapinit# Linux and MacOSpython3-mnovowrapinitUsageCommand line:exclamation: In Linux and macOS, Python2 ispython2and Python3 ispython3.  However,
in Windows, Python3 is calledpython, too. Please notice the difference.Show help information of each module# Windowspython-mnovowrap.assembly-h
python-mnovowrap.validate-h
python-mnovowrap.merge-h# Linux and MacOSpython3-mnovowrap.assembly-h
python3-mnovowrap.validate-h
python3-mnovowrap.merge-hAssembly and validate# Windows, single samplepython-mnovowrap-input[input1][input2]-taxon[taxonomy]# Windows, batch mode for numerous samplespython-mnovowrap-list[listfile]# Linux and MacOS, single samplepython3-mnovowrap-input[input1][input2]-taxon[taxonomy]# Linux and MacOS, batch mode for numerous samplespython3-mnovowrap-list[listfile]Only validate# Windowspython-mnovowrap.validate-input[inputfile]-taxon[taxonomy]# Linux and MacOSpython3-mnovowrap.validate-input[inputfile]-taxon[taxonomy]Only merge# Windowspython-mnovowrap.merge-input[inputfile]# Linux and MacOSpython3-mnovowrap.merge-input[inputfile]Graphical user interfaceIf installed with pip,# Windowspython-mnovowrap# Linux and MacOSpython3-mnovowrapIf use the portable version, just double-click thenovowrap.exeornovowrapin
the folder.Then click the button to choose which module to use. Notice that if one of the
option was set to the wrong value, the program will refuse to run and hint the
user to correct the invalid option.InputTheassemblymodule acceptsgzorfastqformat as input. If useinput list, the list file should becsvformat. If usereferencefile instead
of automatically get from NCBI, the file format should begenbank.Themergemodule acceptsfastaformat as input.Thevalidatemodule acceptsfastaformat as input. If usereferencefile
instead of automatically get from NCBI, the file format should begenbankorfastaas long as it is a complete chloroplast genome.Output.gbfiles:genbankformat sequence, with annotation of the boundary of
LSC/SSC/IR regions..rotatefiles: rotated sequence asfastaformat; start fromtrnH-psbA, same direction with
reference.pdffiles: figure of validation of assembly_RC_files: if filenames contain_RC_, it means one of the regions of the
sequence was adjusted according to the reference. The unadjusted sequence
could be found inTempfolder.OptionsAssemblyGeneralThese options are for general usage.-hor-help: print help message-input [filenames]: input filenames, could be single or pair-end, support gz
and fastq format-list [filenames]: input list for batch mode. The list should be a csv file
with three columns,Input 1,Input 2,TaxonomyIf only has one input file, just leave theInput 2column empty.Please usefull pathof file names, for instance,d:\data\sample-1\forward.fastqinstead offorward.fastqorsample-1\forward.fastq.-ref [filename]: reference file for assembly and validation, should begenbankformat containsonly onechloroplast genome sequence. Extra
sequences will beignored. For automatic running,-taxonis recommended-taxon [taxonomy name]: taxonomy name of the sample, space is allowed.  For
instance,-taxon Oryza sativa, will find reference chloroplast genome of_Oryza sativa_from NCBI RefSeq database. If not found, will find most related
species' reference,Oryza,Poaceae,Poaleset al.If-refand-taxonwere both not set, will use_Nicotiana tabacum_to get the reference (which is one of the earliest sequenced chloroplast
genomes)-out [folder name]: output folder, if not set, the program will auto
create it according to the input file's nameAdvancedThese options are for advanced users. If not sure, just keep the default
value.-platform [illumina/ion]: sequencing platform, the default isillumina. If
use ion-torrent, set-platform ion-insert_size [number]: the insert size of the sequencing library, should be
integer-seed [names]: gene names as seeds for assembly, separated by commas, the
default seeds arerbcL,psaB,psaC,rrn23-seed_file [filename]: seed file, will overwrite-seedoption-split [number]: split input file, only use[number]of them, useful for
large data while computer memory is limited.  For instance,-split 10000000will only use 10 million reads-kmer [number]: kmer size for assembly, should be an odd number. Most time
it's unnecessary to change it-min [number]: minimum genome size, default is 100 kB-max [number]: maximum genome size, default is 200 kB. Only change-minand-maxif the target genome size is out of the default range. The program
needn't know the precise size of the genome-mem [number]: memory limit, the unit is GB. For instance,-mem 8will
limit the memory usage to 8 GB. Should be integer-perc_identity [number]: the threshold of minimum percent of identity, used
for validation with BLAST. The default value is0.7. Should be a float number
between 0 and 1.-len_diff [number]: the threshold of maximum percent of length different of
query and reference. Used for eliminating invalid assembly results. If the
sequence length's difference of assembly and reference genome is larger than
the value, the assembly result will be discarded. The default value is0.2.
Should be a float number between 0 and 1.-debug: print debug information if set-mt: for mitochondria genomes (experimental function)-simple_validate: for chloroplast genomes without quadripartite structureValidateGeneral-hor-help: print help message-input [filename]: input filename. Only supportfastaformat-ref [filename], reference file for assembly and validation, should begenbankorfastaformat that containsonly onechloroplast genome
sequence. Extra sequences will beignored.-taxon [taxonomy name]: taxonomy name of the reference's species, space
is allowed. Recommend to use same genus or family, or higher rank if it's well
known that the target taxonomy's chloroplast genome is conserved.-out [folder name]: output folder, if not set, the program will automatically
create it according to input file's nameAdvanced-perc_identity [number]: the threshold of minimum percent of identity, used
for validation with BLAST. The default value is0.7. Should be a float number
between 0 and 1.-len_diff [number]: the threshold of maximum percent of length different of
query and reference. Used for eliminating invalid assembly results. If the
sequence length's difference of assembly and reference genome is larger than
the value, the assembly result will be discarded. The default value is0.2.
Should be a float number between 0 and 1.-debug: print debug information if set-mt: for mitochondria genomes (experimental function)-simple_validate: for chloroplast genomes without quadripartite structureMerge-hor-help: print help message-input [filename]: input filename. Only supportfastaformat-out [folder name]: output folder, if not set, the program will auto create
it according to the input file's namePerformanceThe most time-consuming step is assembly. If the chloroplast genome's reads in
sequencing data is plentiful enough, and the computer's memory is big enough
for the data size, the assembly will be finished in minutes.The validation step usually could finish in less than one minute. If slower,
please check the Internet connection since the program may query the NCBI
database.The merge module could cost seconds or minutes. It depends on input data.
The complex relationship of contigs requires much more time.CitationWu, P., Xu, C., Chen, H., Yang, J., Zhang, X. and Zhou, S. (2021), NOVOWrap: An automated solution for plastid genome assembly and structure standardization. Molecular Ecology Resources.https://doi.org/10.1111/1755-0998.13410LicenseThe software itself is licensed underAGPL-3.0(not include third-party
software).Q&APlease submit your questions in theIssuepage :smiley:Q: I can't see the full UI, some part was missing.A: Please try to drag the corner of the window to enlarge it. We got reports
that some users in macOS have this issue.Q: I got the error message that the program failed to install
perl/BLAST/NOVOPlasty.A: Uncommonly, users in a specific area will have connection issue for those
websites. Users have to manually download packages and install (seeSoftwarefor the download links).For Windows users, please download and unpack files into%HOMEDRIVE%%HOMEPATH%/.novowrap.For Linux  and MacOS users, please download and unpack files into~/.novowrap.Q: I got the error message that I don't havetkintermodule installed.A: If you want to run GUI on Linux or macOS, this error may happen because the
Python you used did not include tkinter as default package (kind of weird). Run# Debian and Ubuntu
sudo apt install python3-tk
# CentOS
sudo yum install python3-tkmay help.For macOS users or linux users without root privilege, please try to install the
newest version of Python or to use conda, seecondaandPythonfor details.Q: It says my input is invalid, but I'm sure it's OK!A: Please check your files' path. Thespacecharacter in the folder name
or filename may cause this error.Q: It saysImportError: Bio.Alphabet has been removed from Biopythonand
the program failed to start.A: In2020.9, Biopython removed Bio.Alphabet module inv1.78, which may cause this trouble
in the old version of novowrap. Please upgrade yournovowraptov0.97or higher. If you find difficult to upgrade novowrap, please try to use the
portable packages.Q: I want to assemble mitochondria genomes.A: add-mtoption or click the related checkbutton on the GUI.
Since mitochondria genomes do not have a stable
and uniform structure like chloroplast, wet-lab experiments may be necessary
for verification.Q: I want to assemble chloroplast genomes without quadripartite structure.A: add-simple_validateoption in command-line or click the related checkbutton
on the GUI. Note that without quadripartite structure, the Validate module
will skip the adjustment of the structure of the sequences. We have test this function onCytinus hypocistis, a parasitic plant having a 19kb plastid genome.Q: I am acondauser...A: Installnovowrapwithconda install -c wpwupingwp novowrapand the usage is the same.
In order to avoid potential conflicts with other packages, it is highly recommended
to create a new running environment withcondabefore installation. For example,conda create -n test
conda activate test
conda install -c wpwupingwp novowrapIf fail, try to download conda packages fromthis link,
and runcondainstall[filename]Q: I want to use novowrap in parallel.A: Ideally, each novowrap instance is independent. However, the initialization process
could affect other running instances. To avoid this, please running "python3 -m novowrap init"
one time before running in parallel.Q: The assembly failed.A: Make sure your sequencing data is okay. Otherwise, try to set the "-min" and "-max" options to
restrict the size of the target genome. Sometimes it may fail due to inappropriate size values. The
default size is 100-200 kb, which is okay for the common situation.