\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[ngerman,english]{babel}



\usepackage{float}








\begin{document}

\title{NOVOWrap: An automated solution for plastid genome assembly and
structure standardization}



\author[1]{Ping Wu}%
\author[2]{Hao Chen}%
\author[1]{Chao Xu}%
\author[1]{Jie Yang}%
\author[1]{Xian-chun Zhang}%
\author[1]{Shi-Liang Zhou}%
\affil[1]{Institute of Botany Chinese Academy of Sciences}%
\affil[2]{Shaanxi University of Science and Technology}%


\vspace{-1em}



  
  \date{May 23, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Plastid genomes are unique to plants and play an important role in
genomics and evolutionary biology. Next-generation sequencing has
revolutionized plastid genome data acquisition in a way that genome
assembly and annotation became bottlenecks for large plastid genome data
usage. Here we develop a novel open-source, cross-platform tool,
NOVOWrap, with both command-line and graphical user interfaces for
plastid genome automatic assembly using personal computers. With minimum
inputs and user intervention, NOVOWrap could automatically assemble
plastid genomes, validate results and standardize the structure with
affordable computer resources. The performance of the software has been
successfully benchmarked against eleven plastid genomes of species
belonging to lycopods, gymnosperms, and angiosperms. The program is
expected to liberate researchers from laborious computer manipulations
and create reliable and standard genomic data.%
\end{abstract}%



\sloppy


Title:

NOVOWrap: An automated solution for plastid genome assembly and
structure standardization

\textbf{Authors:}

Ping Wu\textsuperscript{1,2}, Hao Chen\textsuperscript{3}, Chao
Xu\textsuperscript{1}, Jie Yang\textsuperscript{1,2}, Xianchun
Zhang\textsuperscript{1,2} and Shiliang Zhou\textsuperscript{1,2*}

\textbf{Affiliations:}

\textsuperscript{1}State Key Laboratory of Systematic and Evolutionary
Botany, Institute of Botany, Chinese Academy of Sciences, Beijing
100093, China

\textsuperscript{2}University of Chinese Academy of Sciences, Beijing
100049, China

\textsuperscript{3} Shaanxi University of Science and Technology, Xi'an
710021, China

*To whom correspondence should be addressed.

Abstract

Plastid genomes are unique to plants and play an important role in
genomics and evolutionary biology. Next-generation sequencing has
revolutionized plastid genome data acquisition in a way that genome
assembly and annotation became bottlenecks for large plastid genome data
usage. Here we develop a novel open-source, cross-platform tool,
NOVOWrap, with both command-line and graphical user interfaces for
plastid genome automatic assembly using personal computers. With minimum
inputs and user intervention, NOVOWrap could automatically assemble
plastid genomes, validate results and standardize the structure with
affordable computer resources. The performance of the software has been
successfully benchmarked against eleven plastid genomes of species
belonging to lycopods, gymnosperms, and angiosperms. The program is
expected to liberate researchers from laborious computer manipulations
and create reliable and standard genomic data.

KEYWORDS: plastid genome, assembly, quadripartite structure

Availability

The source code and portable packages for various operating systems are
available at https://github.com/wpwupingwp/novowrap. The software is
released under the AGPL-3.0 license.

Introduction

Plastids (or chloroplasts in green plants) are considered to originate
from a single endosymbiotic event involving an alpha-proteobacterium and
cyanobacterium (Keeling 2010). During the process of evolution, the
genome of the ancient cyanobacterium shrank and became the plastid
genome of approximately 120-160 kb in size (Green 2011).

A typical plastid genome is a circular, double-stranded DNA molecule
with a quadripartite structure, which consists of two inverted repeated
regions (IR), a small single-copy region (SSC) and a large single-copy
region (LSC). The plastid genome usually encodes 110-130 genes with high
homology and collinearity among plant species (Wicke \emph{et al.}2011).

Owing to its conserved structure, moderate sequence variability (Smith
2015), and high copy number in a cell, plastid genomes of partial or
full-length have been widely used in plant phylogeny, comparative
genomics and biotechnology (Tonti-Filippini \emph{et al.} 2017).

The first two plastid genomes were determined in 1986
from\emph{Nicotiana tabacum} (Sugiura \emph{et al.} 1986) and
\emph{Marchantia polymorpha}(Brassell \emph{et al.} 1986). To date,
there are \textasciitilde{}5,000 complete plastid genomes deposited in
GenBank (Sayers \emph{et al.}2019), and the number is soaring since the
advent of next-generation sequencing (NGS) technologies (Twyford \& Ness
2017). Furthermore, several software for annotating plastid genomes have
been developed (Huang \& Cronk 2015; Qu \emph{et al.} 2019; Shi \emph{et
al.} 2019; Tillich \emph{et al.} 2017). Thus, the biggest obstacle to
the data acquisition of plastid genomes seems to be in the assembly
step.

The mainstream strategy to determine a plastid genome is by sequencing
the total DNA including plastid, mitochondrion and nuclear genome
components using NGS technology. Therefore, handling mixed NGS reads is
a challenge in plastid genome assembly. One solution is to map all the
reads to a close related reference genome allowing reads of plastid to
be filtered and collected for assembly. Another solution is to construct
contigs by\emph{de novo} assembly, and plastid related contigs are then
screened and assembled into a complete genome. Because all reads are
used for assembly, this method requires relatively high computing power
that personal computers can hardly provide. Both methods require manual
adjustment of the IR regions to form a complete plastid genome and a
highly similar reference genome is sometimes indispensable (Twyford \&
Ness 2017).

A novel and increasingly popular strategy is to use a universal seed
sequence to bait plastid reads and extend the assemblage cyclically
until the full circle is formed. Such method not only overcomes the
computing burden of processing all the reads, but also obviates the
requirement of complete genomes as a reference (Freudenthal \emph{et
al.} 2019). Two widely used implementations of such strategy are
NOVOPlasty (Dierckxsens \emph{et al.} 2016) and GetOrganelle
(Jin\emph{et al.} 2019). The former hashes all the reads before the
extension step and has its own assembly algorithm instead of calling
SPAdes (Bankevich \emph{et al.} 2012), which requires less running time.

Unfortunately, all available software only have command-line interfaces
and involve complex inputs or settings, which is a challenge for those
who have limited computer skills or knowledge of operating systems
(Attwood \emph{et al.} 2019). Manual intervention to handle the
questionable outputs is also unavoidable. Even NOVOPlasty and
GetOrganelle, which usually generate full-length genome sequences,
produce multiple outputs with opposite directions, different starting
sites, alternate orientation of LSC/SSC, or sometimes mis-assembly.
Moreover, although the commonly used \emph{rbcL} for baiting reads
usually works well, it may fail in some cases such as gene transfer
events or poor quality. Thus, developing more seeds could be helpful
(Lim \emph{et al.} 2018).

Here, we present NOVOWrap, a user-friendly, cross-platform Python
package for plastid genome assembly. The program could work effectively
on a personal computer and generate reliable assembly results with a
standardized structure, with minimal user intervention during the
process. By providing a highly automatic solution, the program could
help to empower researchers with limited bioinformatics skills or
computer resources to more easily determine plastid genomes for
phylogeny, genomics, and biotechnology.

Methods

\emph{Development of new seeds for assembly}

In order to determine the seeds used for assembly, BarcodeFinder
(https://github.com/wpwupingwp/BarcodeFinder) was used to screen plastid
genome sequences of diverse taxa. Briefly, 5,254 plastid genomes of
green plants were downloaded from NCBI RefSeq database (Pruitt \emph{et
al.} 2012). In order to remove redundant data and reduce computation
time, only one genome of each genus was used and 1,906 genomes were
retained. Following this, sequences of each locus were extracted into
multiple files according to annotations. After sequences were aligned
using MAFFT (Katoh \& Standley 2013), sequence polymorphism of each
locus was investigated (Nei 1987; Spellerberg \& Fedor 2003). Finally,
the seeds were determined based on the following five principles:

(1) the sequences of seed are highly conserved; (2) the locus as a seed
should exist and not pseudogenize in most of the taxa; (3) the locus as
a seed should not transfer to the mitochondrial genome or nuclear genome
commonly; (4) the seed candidates should be located far enough from each
other in the plastid genome to prevent insufficient coverage of data or
notorious repeats influencing the assembly start by taking full use of
alternative seeds (for instance, seeds are from different regions of the
quadripartite structure); and (5) since \emph{rbcL} has been recommended
and widely used, it is kept regardless of other principles.

Eventually, \emph{rbcL} , \emph{psaB} , \emph{psaC} and \emph{rrn23}
were selected as candidate seeds (Table 1). The information of other
loci is shown in Supplementary material 1.

\emph{The Assembly module}

For assembly, NOVOWrap accepts both gzip-compressed and uncompressed NGS
data (FASTQ format) as input. Because memory consumption is positively
correlated to data size, oversize files can hardly be handled on normal
personal computers. The Assembly module offers options for extracting
partial data from the original data files to reduce memory usage.

With user-provided taxon information (scientific name or others) and the
assistance of NCBI Taxonomy database, the module downloads the plastid
genome of the most closely related species deposited in NCBI RefSeq
database. Alternatively, the program could run offline if the user
provides the reference locally. Subsequently, the sequences of seeds
were extracted according to the annotations.

With the above discussed inputs (NGS data files and taxonomy) and all
parameters that can be automatically detected by the program (for
instance, read length, pair-end or not, assembly type, and file path of
each input and output), several configuration files using different
seeds are generated for NOVOPlasty.

Next, NOVOPlasty is called by the module. The outputs of NOVOPlasty are
first checked to verify the completeness of the assembly and then are
organized to avoid conflicts between different assembly processes.

To assemble samples, the module could read input information from a
comma separated values file to handle all assemblies automatically. The
three-column CSV file should contain the file names of ``reads 1'' data,
``reads 2'' data (optional, only for pair-end sequencing) and the taxon
information of samples. Owing to the limited memory resources of
personal computers, the program is executed sequentially to complete
batch assembly, although users can also achieve parallelism by opening
multiple programs if the hardware resources are abundant.

\emph{The Validation module}

When the Assembly module finishes running, the Validation module is
internally called. In addition, users could invoke this module manually
by providing the sequence to be validated and a local reference file or
taxon information.

The Validation module uses ``Rotate'' algorithm to adjust the structure
of target and reference sequences and then conducts the collinearity
analysis (Figure 1). It takes several steps:

\begin{enumerate}
\tightlist
\item
  Generate a full-length plastid genome. Extend the target sequence by
  adding a copy to its end. Independently from where the sequence
  starts, there will be one complete genome started with the beginning
  of the LSC region instead of a truncated region.
\item
  Perform self-to-self alignment. Call blastn of BLAST 2.9.0
  (Madden\emph{et al.} 2019) to perform a self-to-self pairwise
  alignment and default options of blastn are used.
\item
  Locate IR regions. Analyze the BLAST output. Find the longest match
  that has at least three copies (four if the sequence does not start
  with one truncated IR region), which should be IR.
\item
  Determine the other regions. According to the boundary of the IRs,
  locate the LSC and SSC regions. Extract a complete plastid genome with
  the order of LSC-IR-SSC-IR ensuring that the starting site of the
  sequence is on the 5' or 3' (if the LSC region is
  reverse-complemented) terminal of the LSC region.
\item
  Adjust the reference. Repeat steps 1 to 4 on the reference sequence.
\item
  Align target and reference sequences. Use BLAST to perform the
  pairwise alignment for adjusted target and reference sequences. While
  the sequence identity threshold of alignment is between 0 and 100\%,
  in this step, the default low threshold (0.7) allows BLAST to ignore
  the mismatch in the middle of alignment and continue to extend the
  alignment. Hence, the alignment process prefers to focus on the
  structure similarity instead of the sequence one.
\item
  Analyze the alignment result. Because the target and reference
  sequences have the same starting site (steps 4 and 5) and the boundary
  of each region is known, regions of two sequences can be easily
  compared. This may have three possible results. If all the sequences
  match but they are in different directions, the direction of the whole
  sequence needs to be altered. If the direction of LSC or SSC is
  inconsistent with the reference, the orientation of the LSC or SSC
  needs to be adjusted. Besides the two cases mentioned above, the
  process treats it as a problematic assembly due to the conservatism of
  the plastid genome.
\item
  Output validated sequences with a standardized structure. After
  adjusting the starting site, direction of the strand, and orientation
  of four major regions, the verified plastid genome with a standardized
  structure is generated. In addition, the results of collinearity
  analysis and unadjusted sequences are also available in case of need.
\item
  Since both the plus and minus strands are considered when finding the
  repeated regions, apart from IR regions, theoretically, the program
  could also recognize direct repeats (DR). For those species that do
  not have a quadripartite structure, the Validation module may not work
  as normal, such as species of \emph{Erodium} (Blazier \emph{et
  al.}2011) and some parasitic plants (Bellot \& Renner 2016).
\end{enumerate}

If the assembly failed to pass the validation, the Assembly module was
recalled to use another seed for assembly. When at least one validated
assembly is found by the Validation module or all seeds are tried, the
program stops. If no validated assembly is generated after trying all
seeds, the Merge module tries to build a complete plastid genome based
on contigs created using different seeds.

\emph{The Merge module}

The Merge module implemented an edited overlap-Layout-consensus
algorithm (Li \emph{et al.} 2012) to merge contigs according to the
overlaps between them. The procedures are as follows:

\begin{enumerate}
\tightlist
\item
  Make a copy. Since the input contigs could be in sense or antisense
  strands, add a reverse-complemented copy of the original input to
  ensure that at least one copy of contigs are all in sense strands.
\item
  Self-to-self alignment. Call blastn of BLAST 2.9.0 to do self-to-self
  alignment with default options.
\item
  Analyze the BLAST output. Only the forward overlapped match is kept,
  that is, the query and subject sequence are both in the sense strand,
  and the match is between the beginning of the downstream sequence and
  the ending of the upstream sequence. In addition, nested overlap is
  omitted to remove short redundant contigs.
\item
  Generate a unidirectional graph. According to the overlap information
  above, a unidirectional graph is generated. The nodes of the graph
  represent the contigs and the directed edges represent overlaps
  between contigs. Since there are two copies of contigs in the opposite
  pattern, ideally, there might be two major circles in the graph.
\item
  Cut edges. The transitively inferable edges, non-branching stretches
  and alternative paths that go through the same node are removed. The
  edges across the two circles are also removed. All removed edges
  represent incorrect overlapping relationships, mainly due to repeated
  sequences, especially IR regions in plastid genomes.
\item
  Extract the full circle found in the graph. According to the overlap
  information, the program merges contigs to generate circular
  sequences.
\end{enumerate}

If the input contigs contain enough head-to-tail overlapping sequences,
a whole plastid genome is likely to be formed and the program may
generate two circular sequences with opposite orientation. Finally, the
Validation module is called to test the output.

Results

\emph{Benchmark with sequencing data}

Ten species were used to benchmark the usability of the software and the
applicability of the seeds. Species representing gymnosperms, basal
angiosperms, monocots, and eudicots were sequenced. \emph{Pycnostachys
reticulate} and \emph{Nepeta stewartiana} are from newly sequenced genus
and another four are newly sequenced species. Additionally, in order to
test the ability of the program to recognize DR structure and to expand
its application to wider taxonomy, \emph{Selaginella sanguinolenta} , a
species of lycopods, was also used.

Purified DNA was acquired from the Plant DNA Bank of China. Sequencing
was carried out at the Huada Genomics Institute (BGI, Shenzhen, China).
Approximately 50 GB of PE-100 sequencing data per sample were collected.
In order to reduce the memory requirement, only 10,000,000 reads of each
sample were used for assembly. NOVOWrap v0.95 was run on a PC with
Microsoft Windows 10 operating system and 32 GB memory to assemble all
data using default options. The reference genomes of each sample were
automatically chosen by the program. The output genomes were
crossverified based on the results of the collinearity analysis. For
further verification, the assemblies of four species, \emph{Cycas
debaoensis} , \emph{Brasenia schreberi} , \emph{Oryza alta}
and\emph{Firmiana simplex,} were aligned to existing plastid genomes
deposited in GenBank using MAFFT v7.409 (Katoh \& Standley 2013).

The assembled sequences were then annotated using Plann (Huang \& Cronk
2015) and GeSeq (Tillich et al. 2017). The start and stop codon of genes
were manually corrected with Unipro UGENE (Okonechnikov \emph{et
al.}2012) if necessary. The annotation results were finally visualized
with OGDRAW (Greiner \emph{et al.} 2019).

The assembled results are listed in Table 2. All samples were
successfully assembled using NOVOWrap v0.95. The memory usage of the
program stabilized at \textasciitilde{}12 GB and the time cost ranged
between 6 and 20 minutes. For the candidate seeds, two samples failed
with the frequently used \emph{rbcL} but succeeded with other seeds.
While \emph{rbcL} , \emph{psaB} and \emph{psaC} have similar success
rates, \emph{rrn23} only succeeded on \emph{Nepeta stewartiana} , one of
the eleven samples. Each seed generated one or two identical results for
each sample, with one's structure adjusted if necessary. The vast
majority of the samples have identical assembly results with different
seeds with the exception of \emph{Pycnostachys reticulate} . In this
case, 140 candidate results were generated, with lengths ranging between
152,466 and 152,520 bp (Supplementary material 2). The genome structures
of all these candidates are relatively conserved compared to the
reference, \emph{Ocimum tenuiflorum} (species in same family) and have
the same size of SSC (17,671 bp). Their LSC ranged between 83,397 and
83,423 bp and IRs ranged between 25,695 and 25,713 bp. The variations in
size are due to the existence of simple tandem repeats, a difficulty
that NGS alone could hardly solve sometimes.

The Validation module successfully recognized the quadripartite regions
of the sequences, including the DR structure of the lycopod species. The
starting site of each sequence was adjusted according to the respective
references. Inverted LSC and SSC regions were detected and adjusted in
the subsequent collinearity analysis. Most newly determined genomes
displayed high collinearity to the references, except for a few gaps in
the alignment. In addition, the IRs of each genome were similar in size
to the references with no obvious expansion or contraction (Figure 2).

The annotation results further verified the reliability of the assembly
results. No gene loss or pseudogenization occurred in most of the
samples. The only exception is \emph{Salvia campanulata} , which has a
premature termination in one side of the SSC flanked by IRa causing
truncated \emph{ycf1} . Moreover, all the boundaries of the
quadripartite regions determined by the Validation module are consistent
with the results of OGDRAW (Supplementary material 3).

According to the alignment results, the four resequenced genomes are
identical to their references, except for a few one-base insertions or
deletions located in mono nucleotide repeats. This is likely due to
sequencing errors of NGS platforms or intraspecific differences given
that the re-sequenced samples and samples existing in GenBank are from
the same species but different individuals. Annotations demonstrate that
such mutations have no effect on coding sequences.

The Merge module is designed to merge contigs originating from each seed
into one complete plastid genome. For the cases in this study, the Merge
module is skipped because all samples are successfully assembled with
one of the seeds. To test the Merge module, several artificially
generated datasets were used (Supplementary material 4). The result
showed that the Merge module generates a complete plastid genome in most
of the datasets.

Usability

NOVOWrap v0.95 provides both a command-line interface and a graphical
user interface. It is a cross-platform and easy to install. In addition,
it offers portable packages for Linux, Microsoft Windows, and MacOS
operating systems. For the installation, all dependent third-party tools
could be automatically installed if necessary. To further improve
user-friendliness, extensive tests have been conducted to improve the
robustness of the software. Moreover, preventive measures have been set
up for factors that may interrupt the running of the program and cause
inconvenience for users, such as Internet accessibility, the validity of
input data, and the permission to access the work path. The program
requires very few inputs to run and uses universal file formats for both
input and output. Finally, NOVOWrap supports remote access of input
files on the same local area network for convenience.

NOVOWrap is designed to be run on a normal personal computer. The
Validation and Merge modules could finish running in minutes, with
negligible consumption of memory and running time. Although the Assembly
module requires much more resources, based on the feedback from
voluntary testers and our own tests, 16 GB of memory is sufficient to
handle approximately 10,000,000 PE100 reads, 8,000,000 PE150 reads, or
3,000,000 PE200 reads, which is sufficient for obtaining complete
plastid genomes in general cases.

Discussion

The \emph{rbcL} gene, the most frequently used seed for plastid genome
assembly, usually works efficiently, but may fail occasionally. Hence,
three novel seeds were introduced in this study. The \emph{psaB}
and\emph{psaC} functioned as well as \emph{rbcL} . Although
\emph{rrn23}only succeeded in the assembly of \emph{Nepeta stewartiana}
, it could be valuable for some extreme cases. Compared with other newly
developed seeds, \emph{rrn23} is very conservative in 1,331 evaluated
genera. The possible cause of the lower success rate may be the location
difference, that \emph{rrn23} is located in the repeated region, while
others are distributed in single-copy regions. For assembly, starting
with IRs might be harder than LSC/SSC, since the former has extra
possible directions to extend the sequences.

In the benchmark, each sample was assembled with at least one seed.
Although identical sequences derived from different seeds can be used as
supporting evidence for validating the assembly, assembly with one seed
is sufficient for practical use, given that more attempts require much
more time. According to the above, the order of using seeds is optimized
to \emph{rbcL} first, then \emph{psaB} , \emph{psaC,} and \emph{rrn23} .
If none of the seeds work, the whole reference plastid genome is used as
a backup seed.

In early time, the plastid genome is considered to have two natural
isomers with SSC having the opposite polarity (Palmer 1983). The author
considered that the inversion is mediated by the IR structure. However,
some recent studies have used inversion as a phylogenetic character
which is thought to be divergent in different lineages. (Walker \emph{et
al.} 2015). Such misinterpretation has deepened, given the fact that
GenBank does not have a mandatory standard for the direction of the SSC.
In this study, both isoforms were found in the assembly results of
multiple samples. In addition, such structural variations are more
likely to be misassembled owing to the existence of IRs (Jin et al.
2019; Wick \emph{et al.} 2015). NOVOWrap automatically adjusts the
orientation of SSC and LSC as the default output. In the case of
potential needs, unchanged sequences are also generated.

Recently, several studies on the structural variation of plastid
genomes, including the abnormal structure of the genome, the expansion
and shrinking of IRs, and the recombination of orders of genes and other
variations have been conducted (Weng \emph{et al.} 2017; Zhu \emph{et
al.} 2016). However, the inconsistent structure of existing data may
hamper further studies. Not only the orientation of SSC/LSC, but also
other features such as different starting sites, inconsistent order of
quadripartite regions and opposite strands of the whole genome, could
cause problems in downstream analysis, such as annotation, alignment,
collinearity analysis and phylogenetic reconstruction. For instance, the
unadjusted sequence of \emph{Salvia campanulata} has been reported to
have drastically shortened IRs, with a length of 3,845 bp. However, the
adjusted sequence showed a normal length of IRs, with a length of 25,535
bp (Supplementary material 3).

Although manual adjustment may solve the problem, the lack of automatic
tools and standards makes it laborious. The Validation module of
NOVOWrap ensures the consistency of assembly results automatically,
liberating users from tedious error-prone manual manipulation. It is
expected to accelerate plastid genome assembly and ease data upload to
public databases with all consistent genome structures.

Acknowledgements

Thanks Yixuan Huang for testing and packaging program in MacOS. We are
grateful to numerous volunteers who have tested the program with
different data and various operating systems on their own personal
computers or workstations.

This work is funded by the Strategic Priority Research Program of
Chinese Academy of Sciences (XDA23080204 and XDA19050303).

Author Contributions

Ping Wu wrote the program and the manuscript. Hao Chen tested and
optimized the code and wrote the user manual. Chao Xu collected the
data. Jie Yang analyzed data of lycopods and Xianchun Zhang guided the
analysis of DR structure. Shiliang Zhou designed the study and edited
the final manuscript.

Data Accessibility

The assembly results have been uploaded to GenBank. The source code of
the program and the user manual are available
at\emph{https://github.com/wpwupingwp/novowrap}.

References

Attwood TK, Blackford S, Brazas MD, Davies A\emph{, et al.} (2019). A
global perspective on evolving bioinformatics and data science training
needs. \emph{Briefings in Bioinformatics} , 20, 398-404.

Bankevich A, Nurk S, Antipov D, Gurevich AA\emph{, et al.} (2012)
SPAdes: a new genome assembly algorithm and its applications to
single-cell sequencing. \emph{Journal of Computational Biology, 19} (5),
455-477. doi:10.1089/cmb.2012.0021

Bellot S, Renner SS. (2016). The plastomes of two species in the
endoparasite genus pilostyles (apodanthaceae) each retain just five or
six possibly functional genes. \emph{Genome Biol Evol} , 8, 189-201.

Blazier CC, Guisinger MM, Jansen RK. (2011). Recent loss of
plastid-encoded ndh genes within Erodium (Geraniaceae). \emph{Plant
Molecular Biology} , 76, 263-272.

Brassell SC, Eglinton G, Marlowe IT, Pflaumann U\emph{, et al.} (1986).
Chloroplast gene organization deduced from complete sequence of
liverwort Marchantia polymorpha chloroplast DNA. \emph{Nature} , 320,
129-133.

Dierckxsens N, Mardulyn P, Smits G. (2016). NOVOPlasty: De novo assembly
of organelle genomes from whole genome data. \emph{Nucleic Acids
Research} , 45.

Freudenthal JA, Pfaff S, Terhoeven N, Korte A\emph{, et al.} (2019) The
landscape of chloroplast genome assembly tools. \emph{bioRxiv} .
doi:https://doi.org/10.1101/665869

Green BR (2011) Chloroplast genomes of photosynthetic
eukaryotes.\emph{Plant Journal, 66} , 34-44.
doi:10.1111/j.1365-313X.2011.04541.x

Greiner S, Lehwark P, Bock R (2019) OrganellarGenomeDRAW (OGDRAW)
version 1.3.1: expanded toolkit for the graphical visualization of
organellar genomes. \emph{Nucleic Acids Research, 47} (W1), W59-W64.
doi:10.1093/nar/gkz238

Huang DI, Cronk QC (2015) Plann: A command-line application for
annotating plastome sequences. \emph{Applications in Plant Sciences, 3}
(8). doi:10.3732/apps.1500026

Jin J, Yu W, Yang J, Song Y\emph{, et al.} (2019) GetOrganelle: a fast
and versatile toolkit for accurate de novo assembly of organelle
genomes. \emph{bioRxiv} . doi:https://doi.org/10.1101/256479

Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software
version 7: improvements in performance and usability. \emph{Molecular
Biology and Evolution, 30} (4), 772-780. doi:10.1093/molbev/mst010

Keeling PJ (2010) The endosymbiotic origin, diversification and fate of
plastids. \emph{Philosophical transactions of the Royal Society of
London. Series B, Biological sciences, 365} , 729-748.
doi:10.1098/rstb.2009.0103

Li Z, Chen Y, Mu D, Yuan J\emph{, et al.} (2012). Comparison of the two
major classes of assembly algorithms: Overlap-layout-consensus and
de-bruijn-graph. \emph{Briefings in Functional Genomics} , 11, 25-37.

Lim CE, Kim GB, Ryu SA, Yu HJ\emph{, et al.} (2018). The complete
chloroplast genome of Artemisia hallaisanensis Nakai (Asteraceae), an
endemic medicinal herb in Korea. \emph{Mitochondrial DNA Part B:
Resources} , 3, 359-360.

Madden TL, Busby B, Ye J. (2019). Reply to the paper : Misunderstood
parameters of NCBI BLAST impacts the correctness of bioinformatics
workflows.1-2.

Nei M. (1987). \emph{Molecular Evolutionary Genetics} : Columbia
University Press.

Okonechnikov K, Golosova O, Fursov M, team U (2012) Unipro UGENE: a
unified bioinformatics toolkit. \emph{Bioinformatics, 28} (8),
1166-1167. doi:10.1093/bioinformatics/bts091

Palmer JD. (1983). Chloroplast DNA exists in two
orientations.\emph{Nature} , 301, 92-93.

Pruitt KD, Tatusova T, Brown GR, Maglott DR. (2012). NCBI Reference
Sequences (RefSeq): Current status, new features and genome annotation
policy. \emph{Nucleic Acids Research} , 40, 130-135.

Qu XJ, Moore MJ, Li DZ, Yi TS. (2019). PGA: A software package for
rapid, accurate, and flexible batch annotation of plastomes. \emph{Plant
Methods} , 15, 1-12.

Sayers EW, Cavanaugh M, Clark K, Ostell J\emph{, et al.} (2019)
GenBank.\emph{Nucleic Acids Research, 47} (D1), D94-D99.
doi:10.1093/nar/gky989

Shi L, Chen H, Jiang M, Wang L\emph{, et al.} (2019). CPGAVAS2, an
integrated plastome sequence annotator and analyzer. \emph{Nucleic Acids
Research} , 47, W65-W73.

Smith DR (2015) Mutation rates in plastid genomes: They are lower than
you might think. \emph{Genome Biol Evol, 7} , 1227-1234.
doi:10.1093/gbe/evv069

Spellerberg IF, Fedor PJ. (2003). A tribute to Claude-Shannon
(1916-2001) and a plea for more rigorous use of species richness,
species diversity and the 'Shannon-Wiener' Index. \emph{Global Ecology
and Biogeography} , 12, 177-179.

Sugiura M, Shinozaki K, Zaita N, Kusuda M\emph{, et al.} (1986). Clone
bank of the tobacco (Nicotiana tabacum) chloroplast genome as a set of
overlapping restriction endonuclease fragments: mapping of eleven
ribosomal protein genes. \emph{Plant Science} , 44, 211-217.

Tillich M, Lehwark P, Pellizzer T, Ulbricht-Jones ES\emph{, et
al.}(2017). GeSeq - Versatile and accurate annotation of organelle
genomes.\emph{Nucleic Acids Research} , 45, W6-W11.

Tonti-Filippini J, Nevill PG, Dixon K, Small I (2017) What can we do
with 1000 plastid genomes? \emph{Plant Journal, 90} , 808-818.
doi:10.1111/tpj.13491

Twyford AD, Ness RW. (2017). Strategies for complete plastid genome
sequencing. \emph{Molecular Ecology Resources} , 17, 858-868.

Walker JF, Jansen RK, Zanis MJ, Emery NC. (2015). Sources of inversion
variation in the small single copy (SSC) region of chloroplast
genomes.\emph{American Journal of Botany} , 102, 1751-1752.

Weng ML, Ruhlman TA, Jansen RK. (2017). Expansion of inverted repeat
does not decrease substitution rates in Pelargonium plastid
genomes.\emph{New Phytologist} , 214, 842-851.

Wick RR, Schultz MB, Zobel J, Holt KE. (2015). Bandage: Interactive
visualization of de novo genome assemblies. \emph{Bioinformatics} , 31,
3350-3352.

Wicke S, Schneeweiss GM, dePamphilis CW, M\selectlanguage{ngerman}üller KF\emph{, et al.} (2011)
The evolution of the plastid chromosome in land plants: Gene content,
gene order, gene function. \emph{Plant Molecular Biology, 76} , 273-297.
doi:10.1007/s11103-011-9762-4

Zhu A, Guo W, Gupta S, Fan W\emph{, et al.} (2016). Evolutionary
dynamics of the plastid inverted repeat: The effects of expansion,
contraction, and loss on substitution rates. \emph{New Phytologist} ,
209, 1747-1756.

Figure legends

\textbf{Figure 1. Schematic diagram of the Rotate algorithm.} In order
to avoid possible interference of truncated region at the starting site,
the original sequence was firstly duplicated to form a full-length
plastid genome in the middle of the sequence. With the self-to-self
comparison with BLAST, the longest repeated region with the highest
number should be the IRs. Subsequently, according to the location of
IRs, the boundaries of LSC and SSC were determined. Finally, full-length
plastid genomes were extracted from the extended sequences and the
collinearity analysis was performed.

Figure 2. Collinearity analysis of the assembly results. A) plastid
genomes with DR structure; B) assembly has inverted LSC compared with
the reference; C) normal comparison result; D) inverted SSC. Red strips
represent the collinearity between the reference and the plus strand of
the assembly, and green strips represent the minus strand. Blank strips
indicate the mismatch of sequences. Crossed strips could be the matches
between IRs in both strands, or indicate the presence of inverted
region, such as B.

Figure\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1. Schematic diagram of the Rotate algorithm}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2. Collinearity analysis of the assembly results}

\textbf{Hosted file}

\verb`Table 1. The sequence polymorphism of selected seeds.docx`  available at \url{https://authorea.com/users/308401/articles/479879-novowrap-an-automated-solution-for-plastid-genome-assembly-and-structure-standardization}

\textbf{Hosted file}

\verb`Table 2. Samples information and the assembly results.docx`  available at \url{https://authorea.com/users/308401/articles/479879-novowrap-an-automated-solution-for-plastid-genome-assembly-and-structure-standardization}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-2/Figure-2}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

