\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[english]{babel}



\usepackage{float}








\begin{document}

\title{A novel workflow to improve multi-locus amplicon genotyping of wildlife
species: an experimental set-up with a known model system}



\author[1]{Mark Gillingham}%
\author[2]{B. Karina Montero}%
\author[3]{Kerstin Wihelm}%
\author[1]{Kara Grudzus}%
\author[3]{Simone Sommer}%
\author[3]{Pablo Santos}%
\affil[1]{Universitat Ulm}%
\affil[2]{Ulm University}%
\affil[3]{University of Ulm}%


\vspace{-1em}



  
  \date{June 18, 2024}


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





\selectlanguage{english}
\begin{abstract}
Genotyping novel complex multigene families is particularly challenging
in non-model organisms. Target primers frequently amplify simultaneously
multiple loci leading to high PCR and sequencing artefacts such as
chimeras and allele amplification bias. Most genotyping pipelines have
been validated in non-model systems whereby the real genotype is unknown
and the generation of artefacts may be highly repeatable. Further
hindering accurate genotyping, the relationship between artefacts and
genotype complexity (i.e. number of alleles per genotype) within a PCR
remains poorly described. Here we investigated the latter by
experimentally combining multiple known major histocompatibility complex
(MHC) haplotypes of a model organism (chicken,
\textbackslash{}textit\{Gallus gallus\}, 43 artificial genotypes with
2-13 alleles per amplicon). In addition to well defined ``optimal''
primers, we simulated a non-model species situation by designing
``cross-species'' primers, with sequence data from closely related
Galliforme species. We applied a novel open-source genotyping pipeline
(ACACIA; \textbackslash{}url\{https://gitlab.com/psc\_santos/ACACIA\}),
and compared its performance with another, previously published pipeline
(AmpliSAS). Allele calling accuracy was higher when using ACACIA
(98.5\textbackslash{}\% vs 97\textbackslash{}\% and
77.8\textbackslash{}\% vs 75.2\textbackslash{}\% for the ``optimal'' and
``cross-species'' datasets respectively). Systematic allele dropout of
three alleles owing to primer mismatch in the ``cross-species'' dataset
explained high allele calling repeatability (100\textbackslash{}\% when
using ACACIA) despite low accuracy, demonstrating that repeatability can
be misleading when evaluating genotyping workflows. Genotype complexity
was positively associated with non-chimeric artefacts, chimeric
artefacts (nonlinearly by leveling when amplifying more than 4-6
alleles) and allele amplification bias. Our study exemplifies and
demonstrates pitfalls researchers should avoid to reliably genotype
complex multigene families.%
\end{abstract}%



\sloppy


\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/NewFigure1/NewFigure1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/NewFigure2/NewFigure2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/NewFigure3/NewFigure3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/NewFigure4/NewFigure4}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/NewFigure5/NewFigure5}

\end{center}
\end{figure}

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

