minimap2 0.2.22.0 → 0.2.24.1

data/ext/minimap2/tex/minimap2.tex

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+\documentclass{bioinfo}
+\copyrightyear{2018}
+\pubyear{2018}
+
+\usepackage{graphicx}
+\usepackage{hyperref}
+\usepackage{url}
+\usepackage{amsmath}
+\usepackage[ruled,vlined]{algorithm2e}
+\newcommand\mycommfont[1]{\footnotesize\rmfamily{\it #1}}
+\SetCommentSty{mycommfont}
+\SetKwComment{Comment}{$\triangleright$\ }{}
+
+\usepackage{natbib}
+\bibliographystyle{apalike}
+
+\DeclareMathOperator*{\argmax}{argmax}
+
+\begin{document}
+\firstpage{1}
+
+\title[Aligning nucleotide sequences with minimap2]{Minimap2: pairwise alignment for nucleotide sequences}
+\author[Li]{Heng Li}
+\address{Broad Institute, 415 Main Street, Cambridge, MA 02142, USA}
+
+\maketitle
+
+\begin{abstract}
+
+\section{Motivation:} Recent advances in sequencing technologies promise
+ultra-long reads of $\sim$100 kilo bases (kb) in average, full-length mRNA or
+cDNA reads in high throughput and genomic contigs over 100 mega bases (Mb) in
+length. Existing alignment programs are unable or inefficient to process such data
+at scale, which presses for the development of new alignment algorithms.
+
+\section{Results:} Minimap2 is a general-purpose alignment program to map DNA or long
+mRNA sequences against a large reference database. It works with accurate short
+reads of $\ge$100bp in length, $\ge$1kb genomic reads at error rate $\sim$15\%,
+full-length noisy Direct RNA or cDNA reads, and assembly contigs or closely
+related full chromosomes of hundreds of megabases in length. Minimap2 does
+split-read alignment, employs concave gap cost for long insertions and
+deletions (INDELs) and introduces new heuristics to reduce spurious alignments.
+It is 3--4 times as fast as mainstream short-read mappers at comparable
+accuracy, and is $\ge$30 times faster than long-read genomic or cDNA
+mappers at higher accuracy, surpassing most aligners specialized in one type of
+alignment.
+
+\section{Availability and implementation:}
+\href{https://github.com/lh3/minimap2}{https://github.com/lh3/minimap2}
+
+\section{Contact:} hengli@broadinstitute.org
+\end{abstract}
+
+\section{Introduction}
+
+Single Molecule Real-Time (SMRT) sequencing technology and Oxford Nanopore
+technologies (ONT) produce reads over 10kbp in length at an error rate
+$\sim$15\%. Several aligners have been developed for such
+data~\citep{Chaisson:2012aa,Li:2013aa,Liu:2016ab,Sovic:2016aa,Liu:2017aa,Lin:2017aa,Sedlazeck169557}.
+Most of them were five times as slow as mainstream short-read
+aligners~\citep{Langmead:2012fk,Li:2013aa} in terms of the number of bases
+mapped per second. We speculated there could be substantial room for speedup on
+the thought that 10kb long sequences should be easier to map than 100bp reads
+because we can more effectively skip repetitive regions, which are often the
+bottleneck of short-read alignment. We confirmed our speculation by achieving
+approximate mapping 50 times faster than BWA-MEM~\citep{Li:2016aa}.
+\citet{Suzuki:2018aa} extended our work with a fast and novel algorithm on
+generating base-level alignment, which in turn inspired us to develop minimap2
+with added functionality.
+
+Both SMRT and ONT have been applied to the sequencing of spliced mRNAs (RNA-seq). While
+traditional mRNA aligners work~\citep{Wu:2005vn,Iwata:2012aa}, they are not
+optimized for long noisy sequence reads and are tens of times slower than
+dedicated long-read aligners. When developing minimap2 initially for aligning
+genomic DNA only, we realized minor modifications could enable the base
+algorithm to map mRNAs as well. Minimap2 becomes a first RNA-seq aligner
+specifically designed for long noisy reads. We have also extended the original
+algorithm to map short reads at a speed faster than several mainstream
+short-read mappers.
+
+In this article, we will describe the minimap2 algorithm and its applications
+to different types of input sequences. We will evaluate the performance and
+accuracy of minimap2 on several simulated and real data sets and demonstrate
+the versatility of minimap2.
+
+\begin{methods}
+\section{Methods}
+
+Minimap2 follows a typical seed-chain-align procedure as is used by most
+full-genome aligners. It collects minimizers~\citep{Roberts:2004fv} of the
+reference sequences and indexes them in a hash table, with the key being the
+hash of a minimizer and the value being a list of locations of the minimizer
+copies. Then for each query
+sequence, minimap2 takes query minimizers as \emph{seeds}, finds exact matches
+(i.e. \emph{anchors}) to the reference, and identifies sets of colinear anchors as
+\emph{chains}. If base-level alignment is requested, minimap2 applies dynamic
+programming (DP) to extend from the ends of chains and to close
+regions between adjacent anchors in chains.
+
+Minimap2 uses indexing and seeding algorithms similar to
+minimap~\citep{Li:2016aa}, and furthers the predecessor with more accurate
+chaining, the ability to produce base-level alignment and the support of
+spliced alignment.
+
+\subsection{Chaining}
+
+\subsubsection{Chaining}
+An \emph{anchor} is a 3-tuple $(x,y,w)$, indicating interval $[x-w+1,x]$ on the
+reference matching interval $[y-w+1,y]$ on the query. Given a list of anchors
+sorted by ending reference position $x$, let $f(i)$ be the maximal chaining
+score up to the $i$-th anchor in the list. $f(i)$ can be calculated with
+dynamic programming:
+\begin{equation}\label{eq:chain}
+f(i)=\max\big\{\max_{i>j\ge 1} \{ f(j)+\alpha(j,i)-\beta(j,i) \},w_i\big\}
+\end{equation}
+where $\alpha(j,i)=\min\big\{\min\{y_i-y_j,x_i-x_j\},w_i\big\}$ is the number of
+matching bases between the two anchors. $\beta(j,i)>0$ is the gap cost. It
+equals $\infty$ if $y_j\ge y_i$ or $\max\{y_i-y_j,x_i-x_j\}>G$ (i.e. the
+distance between two anchors is too large); otherwise
+\begin{equation}\label{eq:chain-gap}
+\beta(j,i)=\gamma_c\big((y_i-y_j)-(x_i-x_j)\big)
+\end{equation}
+In implementation, a gap of length $l$ costs
+\[
+\gamma_c(l)=\left\{\begin{array}{ll}
+0.01\cdot \bar{w}\cdot|l|+0.5\log_2|l| & (l\not=0) \\
+0 & (l=0)
+\end{array}\right.
+\]
+where $\bar{w}$ is the average seed length. For $N$ anchors, directly computing all $f(\cdot)$ with
+Eq.~(\ref{eq:chain}) takes $O(N^2)$ time. Although theoretically faster
+chaining algorithms exist~\citep{Abouelhoda:2005aa}, they
+are inapplicable to generic gap cost, complex to implement and usually
+associated with a large constant. We introduced a simple heuristic to
+accelerate chaining.
+
+We note that if anchor $i$ is chained to $j$, chaining $i$ to a predecessor
+of $j$ is likely to yield a lower score. When evaluating Eq.~(\ref{eq:chain}),
+we start from anchor $i-1$ and stop the process if we cannot find a better
+score after up to $h$ iterations. This approach reduces the average time to
+$O(hN)$. In practice, we can almost always find the optimal chain with
+$h=50$; even if the heuristic fails, the optimal chain is often close.
+
+\subsubsection{Backtracking}
+Let $P(i)$ be the index of the best predecessor of anchor $i$. It equals 0 if
+$f(i)=w_i$ or $\argmax_j\{f(j)+\alpha(j,i)-\beta(j,i)\}$ otherwise. For each
+anchor $i$ in the descending order of $f(i)$, we apply $P(\cdot)$ repeatedly to
+find its predecessor and mark each visited $i$ as `used', until $P(i)=0$ or we
+reach an already `used' $i$. This way we find all chains with no anchors used
+in more than one chains.
+
+\subsubsection{Identifying primary chains}\label{sec:primary}
+In the absence of copy number changes, each query segment should not be mapped
+to two places in the reference. However, chains found at the previous step may
+have significant or complete overlaps due to repeats in the reference~\citep{Li:2010fk}.
+Minimap2 used the following procedure to identify \emph{primary chains} that do
+not greatly overlap on the query.
+
+Let $Q$ be an empty set initially. For each
+chain from the best to the worst according to their chaining scores: if on the
+query, the chain overlaps with a chain in $Q$ by 50\% or higher percentage of
+the shorter chain, mark the chain as secondary to the chain in $Q$; otherwise,
+add the chain to $Q$. In the end, $Q$ contains all the primary chains. We did
+not choose a more sophisticated data structure (e.g. range tree or k-d tree)
+because this step is not the performance bottleneck.
+
+For each primary chain, minimap2 estimates its mapping quality with an
+empirical formula:
+\[
+{\rm mapQ}=40\cdot (1-f_2/f_1)\cdot\min\{1,m/10\}\cdot\log f_1
+\]
+where $\log$ denotes natural logarithm, $m$ is the number of anchors on the primary chain, $f_1$ is the chaining
+score, and $f_2\le f_1$ is the score of the best chain that is secondary to the
+primary chain. Intuitively, a chain is assigned to a higher mapping quality if
+it is long and its best secondary chain is weak.
+
+\subsubsection{Estimating per-base sequence divergence}
+Suppose a query sequence harbors $n$ seeds of length $k$, $m$ of which are
+present in a chain. We want to estimate the sequence divergence $\epsilon$
+between the query and the reference sequences in the chain. This is useful
+when base-level alignment is too expensive to perform.
+
+If we model substitutions with a homogeneous Poisson process along the query
+sequence, the probablity of seeing $k$ consecutive bases without substitutions
+is $e^{-k\epsilon}$. On the assumption that all $k$-mers are independent of
+each other, the likelihood function of $\epsilon$ is
+\[
+\mathcal{L}(\epsilon|n,m,k)=e^{-m\cdot k\epsilon}(1-e^{-k\epsilon})^{n-m}
+\]
+The maximum likelihood estimate of $\epsilon$ is
+\[
+\hat{\epsilon}=\frac{1}{k}\log\frac{n}{m}
+\]
+In reality, sequencing errors are sometimes clustered and $k$-mers are not
+independent of each other, especially when we take minimizers as seeds. These
+violate the assumptions in the derivation above. As a result, $\hat{\epsilon}$
+is only approximate and can be biased. It also ignores long deletions from the
+reference sequence. In practice, fortunately, $\hat{\epsilon}$ is often close
+to and strongly correlated with the sequence divergence estimated from
+base-level alignments. On the several datasets used in
+Section~\ref{sec:long-genomic}, the Spearman correlation coefficient is around
+$0.9$.
+
+\subsubsection{Indexing with homopolymer compressed $k$-mers}
+SmartDenovo
+(\href{https://github.com/ruanjue/smartdenovo}{https://github.com/ruanjue/smartdenovo};
+J. Ruan, personal communication) indexes reads with homopolymer-compressed (HPC)
+$k$-mers and finds the strategy improves overlap sensitivity for SMRT reads.
+Minimap2 adopts the same heuristic.
+
+The HPC string of a string $s$, denoted by ${\rm HPC}(s)$, is constructed by
+contracting homopolymers in $s$ to a single base.  An HPC $k$-mer of $s$ is a
+$k$-long substring of ${\rm HPC}(s)$. For example, suppose $s={\tt GGATTTTCCA}$,
+${\rm HPC}(s)={\tt GATCA}$ and the first HPC 4-mer is ${\tt GATC}$.
+
+To demonstrate the effectiveness of HPC $k$-mers, we performed read overlapping
+for the example {\it E. coli} SMRT reads from PBcR~\citep{Berlin:2015xy}, using
+different types of $k$-mers. With normal 15bp minimizers per 5bp window,
+minimap2 finds 90.9\% of $\ge$2kb overlaps inferred from the read-to-reference
+alignment. With HPC 19-mers per 5bp window, minimap2 finds 97.4\% of overlaps. It achieves this
+higher sensitivity by indexing 1/3 fewer minimizers, which further helps
+performance. HPC-based indexing reduces the sensitivity for current ONT reads, though.
+
+\subsection{Aligning genomic DNA}\label{sec:genomic}
+
+\subsubsection{Alignment with 2-piece affine gap cost}
+
+Minimap2 performs DP-based global alignment between adjacent anchors in a
+chain. It uses a 2-piece affine gap cost~\citep{Gotoh:1990aa}:
+\begin{equation}\label{eq:2-piece}
+\gamma_a(l)=\min\{q+|l|\cdot e,\tilde{q}+|l|\cdot\tilde{e}\}
+\end{equation}
+Without losing generality, we always assume $q+e<\tilde{q}+\tilde{e}$.
+On the condition that $e>\tilde{e}$, it applies cost $q+|l|\cdot e$ to gaps
+shorter than $\lceil(\tilde{q}-q)/(e-\tilde{e})\rceil$ and applies
+$\tilde{q}+|l|\cdot\tilde{e}$ to longer gaps.  This scheme helps to recover
+longer insertions and deletions~(INDELs).
+
+The equation to compute the optimal alignment under $\gamma_a(\cdot)$ is
+\begin{equation}\label{eq:ae86}
+\left\{\begin{array}{l}
+H_{ij} = \max\{H_{i-1,j-1}+s(i,j),E_{ij},F_{ij},\tilde{E}_{ij},\tilde{F}_{ij}\}\\
+E_{i+1,j}= \max\{H_{ij}-q,E_{ij}\}-e\\
+F_{i,j+1}= \max\{H_{ij}-q,F_{ij}\}-e\\
+\tilde{E}_{i+1,j}= \max\{H_{ij}-\tilde{q},\tilde{E}_{ij}\}-\tilde{e}\\
+\tilde{F}_{i,j+1}= \max\{H_{ij}-\tilde{q},\tilde{F}_{ij}\}-\tilde{e}
+\end{array}\right.
+\end{equation}
+where $s(i,j)$ is the score between the $i$-th reference base and $j$-th query
+base. Eq.~(\ref{eq:ae86}) is a natural extension to the equation under affine
+gap cost~\citep{Gotoh:1982aa,Altschul:1986aa}.
+
+\subsubsection{The Suzuki-Kasahara formulation}
+
+When we allow gaps longer than several hundred base pairs, nucleotide-level
+alignment is much slower than chaining. SSE acceleration is critical to the
+performance of minimap2. Traditional SSE implementations~\citep{Farrar:2007hs}
+based on Eq.~(\ref{eq:ae86}) can achieve 16-way parallelization for short
+sequences, but only 4-way parallelization when the peak alignment score reaches
+32767. Long sequence alignment may exceed this threshold. Inspired by
+\citet{Wu:1996aa} and the following work, \citet{Suzuki:2018aa} proposed a
+difference-based formulation that lifted this limitation.
+In case of 2-piece gap cost, define
+\[
+\left\{\begin{array}{ll}
+u_{ij}\triangleq H_{ij}-H_{i-1,j} & v_{ij}\triangleq H_{ij}-H_{i,j-1} \\
+x_{ij}\triangleq E_{i+1,j}-H_{ij} & \tilde{x}_{ij}\triangleq \tilde{E}_{i+1,j}-H_{ij} \\
+y_{ij}\triangleq F_{i,j+1}-H_{ij} & \tilde{y}_{ij}\triangleq \tilde{F}_{i,j+1}-H_{ij}
+\end{array}\right.
+\]
+We can transform Eq.~(\ref{eq:ae86}) to
+\begin{equation}\label{eq:suzuki}
+\left\{\begin{array}{lll}
+z_{ij}&=&\max\{s(i,j),x_{i-1,j}+v_{i-1,j},y_{i,j-1}+u_{i,j-1},\\
+&&\tilde{x}_{i-1,j}+v_{i-1,j},\tilde{y}_{i,j-1}+u_{i,j-1}\}\\
+u_{ij}&=&z_{ij}-v_{i-1,j}\\
+v_{ij}&=&z_{ij}-u_{i,j-1}\\
+x_{ij}&=&\max\{0,x_{i-1,j}+v_{i-1,j}-z_{ij}+q\}-q-e\\
+y_{ij}&=&\max\{0,y_{i,j-1}+u_{i,j-1}-z_{ij}+q\}-q-e\\
+\tilde{x}_{ij}&=&\max\{0,\tilde{x}_{i-1,j}+v_{i-1,j}-z_{ij}+\tilde{q}\}-\tilde{q}-\tilde{e}\\
+\tilde{y}_{ij}&=&\max\{0,\tilde{y}_{i,j-1}+u_{i,j-1}-z_{ij}+\tilde{q}\}-\tilde{q}-\tilde{e}
+\end{array}\right.
+\end{equation}
+where $z_{ij}$ is a temporary variable that does not need to be stored.
+
+An important property of Eq.~(\ref{eq:suzuki}) is that all values are bounded
+by scoring parameters. To see that,
+\[
+x_{ij}=E_{i+1,j}-H_{ij}=\max\{-q,E_{ij}-H_{ij}\}-e
+\]
+With $E_{ij}\le H_{ij}$, we have
+\[
+-q-e\le x_{ij}\le\max\{-q,0\}-e=-e
+\]
+and similar inequations for $y_{ij}$, $\tilde{x}_{ij}$ and $\tilde{y}_{ij}$.
+In addition,
+\[
+u_{ij}=z_{ij}-v_{i-1,j}\ge\max\{x_{i-1,j},\tilde{x}_{i-1,j}\}\ge-q-e
+\]
+As the maximum value of $z_{ij}=H_{ij}-H_{i-1,j-1}$ is $M$, the maximal
+matching score, we can derive
+\[
+u_{ij}\le M-v_{i-1,j}\le M+q+e
+\]
+In conclusion, in Eq.~(\ref{eq:suzuki}), $x$ and $y$ are bounded by $[-q-e,-e]$,
+$\tilde{x}$ and $\tilde{y}$ by $[-\tilde{q}-\tilde{e},-\tilde{e}]$, and $u$ and
+$v$ by $[-q-e,M+q+e]$. When $-128\le-q-e<M+q+e\le127$, each of them can be stored as
+a 8-bit integer. This enables 16-way SSE vectorization regardless of the peak
+score of the alignment.
+
+For a more efficient SSE implementation, we transform the row-column coordinate
+to the diagonal-antidiagonal coordinate by letting $r\gets i+j$ and $t\gets i$.
+Eq.~(\ref{eq:suzuki}) becomes:
+\begin{equation*}
+\left\{\begin{array}{lll}
+z_{rt}&=&\max\{s(t,r-t),x_{r-1,t-1}+v_{r-1,t-1},y_{r-1,t}\\
+&&+u_{r-1,t},\tilde{x}_{r-1,t-1}+v_{r-1,t-1},\tilde{y}_{r-1,t}+u_{r-1,t}\}\\
+u_{rt}&=&z_{rt}-v_{r-1,t-1}\\
+v_{rt}&=&z_{rt}-u_{r-1,t}\\
+x_{rt}&=&\max\{0,x_{r-1,t-1}+v_{r-1,t-1}-z_{rt}+q\}-q-e\\
+y_{rt}&=&\max\{0,y_{r-1,t}+u_{r-1,t}-z_{rt}+q\}-q-e\\
+\tilde{x}_{rt}&=&\max\{0,\tilde{x}_{r-1,t-1}+v_{r-1,t-1}-z_{rt}+\tilde{q}\}-\tilde{q}-\tilde{e}\\
+\tilde{y}_{rt}&=&\max\{0,\tilde{y}_{r-1,t}+u_{r-1,t}-z_{rt}+\tilde{q}\}-\tilde{q}-\tilde{e}
+\end{array}\right.
+\end{equation*}
+In this formulation, cells with the same diagonal index $r$ are independent of
+each other. This allows us to fully vectorize the computation of all cells on
+the same anti-diagonal in one inner loop. It also simplifies banded alignment (500bp band width by default),
+which would be difficult with striped vectorization~\citep{Farrar:2007hs}.
+
+On the condition that $q+e<\tilde{q}+\tilde{e}$ and $e>\tilde{e}$, the initial
+values in the diagonal-antidiagonal formuation are
+\[
+\left\{\begin{array}{l}
+x_{r-1,-1}=y_{r-1,r}=-q-e\\
+\tilde{x}_{r-1,-1}=\tilde{y}_{r-1,r}=-\tilde{q}-\tilde{e}\\
+u_{r-1,r}=v_{r-1,-1}=\eta(r)\\
+\end{array}\right.
+\]
+where
+\[
+\eta(r)=\left\{\begin{array}{ll}
+-q-e & (r=0) \\
+-e & (r<\lceil\frac{\tilde{q}-q}{e-\tilde{e}}-1\rceil) \\
+r\cdot(e-\tilde{e})-(\tilde{q}-q)-\tilde{e} & (r=\lceil\frac{\tilde{q}-q}{e-\tilde{e}}-1\rceil) \\
+-\tilde{e} & (r>\lceil\frac{\tilde{q}-q}{e-\tilde{e}}-1\rceil)
+\end{array}\right.
+\]
+These can be derived from the initial values for Eq.~(\ref{eq:ae86}).
+
+When performing global alignment, we do not need to compute $H_{rt}$ in each cell.
+We use 16-way vectorization throughout the alignment process. When extending
+alignments from ends of chains, we need to find the cell $(r,t)$ where $H_{rt}$
+reaches the maximum. We resort to 4-way vectorization to compute
+$H_{rt}=H_{r-1,t}+u_{rt}$. Because this computation is simple,
+Eq.~(\ref{eq:suzuki}) is still the dominant performance bottleneck.
+
+In practice, our 16-way vectorized implementation of global alignment is three
+times as fast as Parasail's 4-way vectorization~\citep{Daily:2016aa}.  Without
+banding, our implementation is slower than Edlib~\citep{Sosic:2017aa}, but with
+a 1000bp band, it is considerably faster. When performing global alignment
+between anchors, we expect the alignment to stay close to the diagonal of the
+DP matrix. Banding is applicable most of the time.
+
+\subsubsection{The Z-drop heuristic}
+
+With global alignment, minimap2 may force to align unrelated sequences between
+two adjacent anchors. To avoid such an artifact, we compute accumulative
+alignment score along the alignment path and break the alignment where the
+score drops too fast in the diagonal direction. More precisely, let $S(i,j)$ be
+the alignment score along the alignment path ending at cell $(i,j)$ in the DP
+matrix. We break the alignment if there exist $(i',j')$ and $(i,j)$, $i'<i$ and
+$j'<j$, such that
+\[
+S(i',j')-S(i,j)>Z+e\cdot|(i-i')-(j-j')|
+\]
+where $e$ is the gap extension cost and $Z$ is an arbitrary threshold.
+This strategy is first used in BWA-MEM. It is similar to X-drop employed in
+BLAST~\citep{Altschul:1997vn}, but unlike X-drop, it would not break the
+alignment in the presence of a single long gap.
+
+When minimap2 breaks a global alignment between two anchors, it performs local
+alignment between the two subsequences involved in the global alignment, but
+this time with the one subsequence reverse complemented. This additional
+alignment step may identify short inversions that are missed during chaining.
+
+\subsubsection{Filtering out misplaced anchors}
+Due to sequencing errors and local homology, some anchors in a chain may be
+wrong. If we blindly align regions between two misplaced anchors, we will
+produce a suboptimal alignment. To reduce this artifact, we filter out
+anchors that lead to a $>$10bp insertion and a $>$10bp deletion at the same
+time, and filter out terminal anchors that lead to a long gap towards the ends
+of a chain. These heuristics greatly alleviate the issues with misplaced
+anchors, but they are unable to fix all such errors. Local misalignment is a
+limitation of minimap2 which we hope to address in future.
+
+\subsection{Aligning spliced sequences}
+
+The algorithm described above can be adapted to spliced alignment. In this
+mode, the chaining gap cost distinguishes insertions to and deletions from the
+reference: $\gamma_c(l)$ in Eq.~(\ref{eq:chain-gap}) takes the form of
+\[
+\gamma_c(l)=\left\{\begin{array}{ll}
+0.01\cdot\bar{w}\cdot l+0.5\log_2 l & (l>0) \\
+\min\{0.01\cdot\bar{w}\cdot|l|,\log_2|l|\} & (l<0)
+\end{array}\right.
+\]
+Similarly, the gap cost function used for DP-based alignment is changed to
+\[
+\gamma_a(l)=\left\{\begin{array}{ll}
+q+l\cdot e & (l>0) \\
+\min\{q+|l|\cdot e,\tilde{q}\} & (l<0)
+\end{array}\right.
+\]
+In alignment, a deletion no shorter than $\lceil(\tilde{q}-q)/e\rceil$ is
+regarded as an intron, which pays no cost to gap extensions.
+
+To pinpoint precise splicing junctions, minimap2 introduces reference-dependent
+cost to penalize non-canonical splicing:
+\begin{equation}\label{eq:splice}
+\left\{\begin{array}{l}
+H_{ij} = \max\{H_{i-1,j-1}+s(i,j),E_{ij},F_{ij},\tilde{E}_{ij}-a(i)\}\\
+E_{i+1,j}= \max\{H_{ij}-q,E_{ij}\}-e\\
+F_{i,j+1}= \max\{H_{ij}-q,F_{ij}\}-e\\
+\tilde{E}_{i+1,j}= \max\{H_{ij}-d(i)-\tilde{q},\tilde{E}_{ij}\}\\
+\end{array}\right.
+\end{equation}
+Let $T$ be the reference sequence. $d(i)$ is computed as
+\[d(i)=\left\{\begin{array}{ll}
+0 & \mbox{if $T[i+1,i+3]$ is ${\tt GTA}$ or ${\tt GTG}$} \\
+p/2 & \mbox{if $T[i+1,i+3]$ is ${\tt GTC}$ or ${\tt GTT}$} \\
+p & \mbox{otherwise}
+\end{array}\right.\]
+where $T[i,j]$ extracts a substring of $T$ between $i$ and $j$ inclusively.
+$d(i)$ penalizes non-canonical donor sites with $p$ and less frequent Eukaryotic
+splicing signal ${\tt GT[C/T]}$ with $p/2$~\citep{Irimia:2008aa}. Similarly,
+\[a(i)=\left\{\begin{array}{ll}
+0 & \mbox{if $T[i-2,i]$ is ${\tt CAG}$ or ${\tt TAG}$} \\
+p/2 & \mbox{if $T[i-2,i]$ is ${\tt AAG}$ or ${\tt GAG}$} \\
+p & \mbox{otherwise}
+\end{array}\right.\]
+models the acceptor signal. Eq.~(\ref{eq:splice}) is close to an equation in
+\citet{Zhang:2006aa} except that we allow insertions immediately followed by
+deletions and vice versa; in addition, we use the Suzuki-Kasahara diagonal
+formulation in actual implementation.
+
+If RNA-seq reads are not sequenced from stranded libraries, the read strand
+relative to the underlying transcript is unknown. By default, minimap2 aligns
+each chain twice, first assuming ${\tt GT}$--${\tt AG}$ as the splicing signal
+and then assuming ${\tt CT}$--${\tt AC}$, the reverse complement of ${\tt
+GT}$--${\tt AG}$, as the splicing signal. The alignment with a higher score is
+taken as the final alignment. This procedure also infers the relative strand of
+reads that span canonical splicing sites.
+
+In the spliced alignment mode, minimap2 further increases the density of
+minimizers and disables banded alignment. Together with the two-round DP-based
+alignment, spliced alignment is several times slower than genomic DNA
+alignment.
+
+\subsection{Aligning short paired-end reads}
+
+During chaining, minimap2 takes a pair of reads as one fragment with a gap of
+unknown length in the middle. It applies a normal gap cost between seeds on the
+same read but is a more permissive gap cost between seeds on different reads.
+More precisely, the gap cost during chaining is ($l\not=0$):
+\[
+\gamma_c(l)=\left\{\begin{array}{ll}
+0.01\cdot\bar{w}\cdot |l|+0.5\log_2 |l| & \mbox{if two seeds on the same read} \\
+\min\{0.01\cdot\bar{w}\cdot|l|,\log_2|l|\} & \mbox{otherwise}
+\end{array}\right.
+\]
+After identifying primary chains (Section~\ref{sec:primary}), we split each
+fragment chain into two read chains and perform alignment for each read as in
+Section~\ref{sec:genomic}.  Finally, we pair hits of each read end to find
+consistent paired-end alignments.
+
+\end{methods}
+
+\section{Results}
+
+Minimap2 is implemented in the C programming language and comes with APIs in
+both C and Python. It is distributed under the MIT license, free to both
+commercial and academic uses. Minimap2 uses the same base algorithm for all
+applications, but it has to apply different sets of parameters depending on
+input data types. Similar to BWA-MEM, minimap2 introduces `presets' that
+modify multiple parameters with a simple invocation. Detailed settings
+and command-line options can be found in the minimap2 manpage. In addition to
+the applications evaluated in the following sections, minimap2 also retains
+minimap's functionality to find overlaps between long reads and to search
+against large multi-species databases such as \emph{nt} from NCBI.
+
+\subsection{Aligning long genomic reads}\label{sec:long-genomic}
+
+\begin{figure}[!tb]
+\centering
+\includegraphics[width=.5\textwidth]{roc-color.pdf}
+\caption{Evaluation on aligning simulated reads. Simulated reads were mapped 
+to the primary assembly of human genome GRCh38. A read is considered correctly
+mapped if its longest alignment overlaps with the true interval, and the
+overlap length is $\ge$10\% of the true interval length.  Read alignments are
+sorted by mapping quality in the descending order. For each mapping quality
+threshold, the fraction of alignments (out of the number of input reads) with
+mapping quality above the threshold and their error rate are
+plotted along the curve. (a) long-read alignment evaluation. 33,088 $\ge$1000bp
+reads were simulated using pbsim~\citep{Ono:2013aa} with error profile sampled
+from file `m131017\_060208\_42213\_*.1.*' downloaded at
+\href{http://bit.ly/chm1p5c3}{http://bit.ly/chm1p5c3}. The N50 read length is
+11,628. Aligners were run under the default setting for SMRT reads.
+Kart outputted all alignments at mapping quality 60, so is not shown in the
+figure. It mapped nearly all reads with 4.1\% of alignments being wrong, less
+accurate than others.  (b) short-read alignment evaluation. 10 million pairs of
+150bp reads were simulated using mason2~\citep{Holtgrewe:2010aa} with option
+`\mbox{--illumina-prob-mismatch-scale 2.5}'. Short-read aligners were run under
+the default setting except for changing the maximum fragment length to
+800bp.}\label{fig:eval}
+\end{figure}
+
+As a sanity check, we evaluated minimap2 on simulated human reads along with
+BLASR~(v1.MC.rc64; \citealp{Chaisson:2012aa}),
+BWA-MEM~(v0.7.15; \citealp{Li:2013aa}),
+GraphMap~(v0.5.2; \citealp{Sovic:2016aa}),
+Kart~(v2.2.5; \citealp{Lin:2017aa}),
+minialign~(v0.5.3; \href{https://github.com/ocxtal/minialign}{https://github.com/ocxtal/minialign}) and
+NGMLR~(v0.2.5; \citealp{Sedlazeck169557}). We excluded rHAT~\citep{Liu:2016ab}
+and LAMSA~\citep{Liu:2017aa} because they either
+crashed or produced malformatted output. In this evaluation, minimap2 has
+higher power to distinguish unique and repetitive hits, and achieves overall
+higher mapping accuracy (Fig.~\ref{fig:eval}a). Minimap2 and
+NGMLR provide better mapping quality estimate: they rarely give repetitive hits
+high mapping quality.  Apparently, other aligners may
+occasionally miss close suboptimal hits and be overconfident in wrong mappings.
+On run time, minimap2 took 200 CPU seconds, comparable to minialign and Kart, and is over
+30 times faster than the rest.  Minimap2 consumed 6.8GB memory at the peak,
+more than BWA-MEM (5.4GB), similar to NGMLR and less than others.
+
+On real human SMRT reads, the relative performance and fraction of mapped reads reported by
+these aligners are broadly similar to the metrics on simulated data. We are
+unable to provide a good estimate of mapping error rate due to the lack of the
+truth.  On ONT $\sim$100kb human reads~\citep{Jain128835}, BWA-MEM failed.
+Kart, minialign and minimap2 are over 70 times faster than others. We have also
+examined tens of $\ge$100bp INDELs in IGV~\citep{Robinson:2011aa} and can
+confirm the observation by~\citet{Sedlazeck169557} that BWA-MEM often breaks
+them into shorter gaps. The issue is much alleviated with minimap2, thanks
+to the 2-piece affine gap cost.
+
+\subsection{Aligning long spliced reads}
+
+We evaluated minimap2 on SIRV control data~(AC:SRR5286959;
+\citealp{Byrne:2017aa}) where the truth is known. Minimap2 predicted 59\,918
+introns from 11\,018 reads. 93.8\% of splice juctions are precise. We examined
+wrongly predicted junctions and found the majority were caused by clustered
+splicing signals (e.g. two adjacent ${\tt GT}$ sites). When INDEL sequencing
+errors are frequent, it is difficult to find precise splicing sites in this
+case. If we allow up to 10bp distance from true splicing sites, 98.4\% of
+aligned introns are approximately correct. It is worth noting that for SIRV, we
+asked minimap2 to model the ${\tt GT..AG}$ splicing signal only without extra
+bases. This is because SIRV does not honor the evolutionarily prevalent signal
+${\tt GT[A/G]..[C/T]AG}$~\citep{Irimia:2008aa}.
+
+\begin{table}[!tb]
+\processtable{Evaluation of junction accuracy on 2D ONT reads}
+{\footnotesize\label{tab:intron}
+\begin{tabular}{p{3.1cm}rrrr}
+\toprule
+& GMAP & minimap2 & SpAln & STAR\\
+\midrule
+Run time (CPU min)        & 631      & 15.9     & 2\,076   & 33.9 \\
+Peak RAM (GByte)          & 8.9      & 14.5     & 3.2      & 29.2\vspace{1em}\\
+\# aligned reads          & 103\,669 & 104\,199 & 103\,711 & 26\,479 \\
+\# chimeric alignments    & 1\,904   & 1\,488   & 0        & 0 \\
+\# non-spliced alignments & 15\,854  & 14\,798  & 17\,033  & 10\,545\vspace{1em}\\
+\# aligned introns        & 692\,275 & 693\,553 & 692\,945 & 78\,603 \\
+\# novel introns          & 11\,239  & 3\,113   & 8\,550   & 1\,214 \\
+\% exact introns          & 83.8\%   & 94.0\%   & 87.9\%   & 55.2\% \\
+\% approx. introns        & 91.8\%   & 96.9\%   & 92.5\%   & 82.4\% \\
+\botrule
+\end{tabular}
+}{Mouse cDNA reads (AC:SRR5286960; R9.4 chemistry) were mapped to the primary assembly of mouse
+genome GRCm38 with the following tools and command options: minimap2 (`-ax
+splice'); GMAP (`-n 0 --min-intronlength 30 --cross-species'); SpAln (`-Q7 -LS
+-S3'); STARlong (according to
+\href{http://bit.ly/star-pb}{http://bit.ly/star-pb}). The alignments were
+compared to the EnsEMBL gene annotation, release 89. A predicted intron
+is \emph{novel} if it has no overlaps with any annotated introns. An intron
+is \emph{exact} if it is identical to an annotated intron. An intron is
+\emph{approximate} if both its 5'- and 3'-end are within 10bp around the ends
+of an annotated intron. Chimeric alignments are defined in the SAM spec~\citep{Li:2009ys}.}
+\end{table}
+
+We next aligned real mouse reads~\citep{Byrne:2017aa} with GMAP~(v2017-06-20;
+\citealp{Wu:2005vn}), minimap2, SpAln~(v2.3.1; \citealp{Iwata:2012aa}) and
+STAR~(v2.5.3a; \citealp{Dobin:2013kx}). In general, minimap2 is more
+consistent with existing annotations (Table~\ref{tab:intron}): it finds
+more junctions with a higher percentage being exactly or approximately correct.
+Minimap2 is over 40 times faster than GMAP and SpAln. While STAR is close to
+minimap2 in speed, it does not work well with noisy reads.
+
+We have also evaluated spliced aligners on a human Nanopore Direct RNA-seq
+dataset (\href{http://bit.ly/na12878ont}{http://bit.ly/na12878ont}). Minimap2
+aligned 10 million reads in $<$1 wall-clock hour using 16 CPU cores. 94.2\% of
+aligned splice junctions consistent with gene annotations. In comparison,
+GMAP under option `-k 14 -n 0 --min-intronlength 30 --cross-species' is 160
+times slower; 68.7\% of GMAP junctions are found in known gene annotations. The
+percentage increases to 84.1\% if an aligned junction within 10bp from an
+annotated junction is considered to be correct. On a public Iso-Seq dataset
+(human Alzheimer brain from
+\href{http://bit.ly/isoseqpub}{http://bit.ly/isoseqpub}), minimap2 is also
+faster at higher junction accuracy in comparison to other aligners in
+Table~\ref{tab:intron}.
+
+We noted that GMAP and SpAln have not been optimized for noisy reads. We are
+showing the best setting we have experimented, but their developers should be
+able to improve their accuracy further.
+
+%\begin{table}[!tb]
+%\processtable{Evaluation of junction accuracy on SMRT Iso-Seq reads}
+%{\footnotesize
+%\begin{tabular}{lrrrr}
+%\toprule
+%                          &      GMAP &  minimap2 &     SpAln &      STAR \\ % one GMAP thread took 14 days to align a tiny fraction of reads
+%\midrule
+%Run time (CPU min)        &         - &       243 &     2,352 &     1,647 \\
+%\# aligned reads          & 1,113,502 & 1,123,025 & 1,094,092 &   682,452 \\
+%\# chimeric alignments    &    48,927 &    33,091 &         0 &         0 \\
+%\# non-spliced alignments &   334,097 &   339,081 &   291,447 &   272,536 \vspace{1em}\\
+%\# aligned introns        & 8,922,221 & 9,071,755 & 9,208,564 & 3,029,121 \\
+%\# novel introns          &    48,927 &    42,773 &    82,230 &    17,791 \\
+%\% exact introns          &    90.6\% &    94.9\% &    91.7\% &    84.7\% \\
+%\% approx. introns        &    94.0\% &    96.9\% &    93.4\% &    93.8\% \\
+%\botrule
+%\end{tabular}
+%}{}
+%\end{table}
+
+\subsection{Aligning short genomic reads}
+
+We evaluated minimap2 along with Bowtie2~(v2.3.3; \citealt{Langmead:2012fk}), BWA-MEM and
+SNAP (v1.0beta23; \citealt{Zaharia:2011aa}). Minimap2 is 3--4 times as fast as Bowtie2 and
+BWA-MEM, but is 1.3 times slower than SNAP. Minimap2 is more accurate on this
+simulated data set than Bowtie2 and SNAP but less accurate than BWA-MEM
+(Fig.~\ref{fig:eval}b). Closer investigation reveals that BWA-MEM achieves
+a higher accuracy partly because it tries to locally align a read in a small
+region close to its mate. If we disable this feature, BWA-MEM becomes slightly
+less accurate than minimap2. We might implement a similar heuristic
+in minimap2 in future.
+
+To evaluate the accuracy of minimap2 on real data, we aligned human reads
+(AC:ERR1341796) with BWA-MEM and minimap2, and called SNPs and small INDELs
+with GATK HaplotypeCaller v3.5~\citep{Depristo:2011vn}. This run was sequenced
+from experimentally mixed CHM1 and CHM13 cell lines. Both of them are homozygous
+across the whole genome and have been \emph{de novo} assembled with SMRT reads
+to high quality. This allowed us to construct an independent truth variant
+dataset~\citep{Li223297} for
+ERR1341796. In this evaluation, minimap2 has higher SNP false negative rate
+(FNR; 2.6\% of minimap2 vs 2.3\% of BWA-MEM), but fewer false positive SNPs per
+million bases (FPPM; 7.0 vs 8.8), similar INDEL FNR (11.2\% vs 11.3\%) and
+similar INDEL FPPM (6.4 vs 6.5). Minimap2 is broadly comparable to BWA-MEM in the
+context of small variant calling.
+
+\subsection{Aligning long-read assemblies}
+
+Minimap2 can align a SMRT assembly (AC:GCA\_001297185.1) against GRCh38 in 7
+minutes using 8 CPU cores, over 20 times faster than nucmer from
+MUMmer4~\citep{Marcais:2018aa}. With the paftools.js script from the minimap2
+package, we called 2.67 million single-base substitutions out of 2.78Gbp
+genomic regions. The transition-to-transversion ratio (ts/tv) is 2.01. In
+comparison, using MUMmer4's dnadiff pipeline, we called 2.86 million
+substitutions in 2.83Gbp at ts/tv=1.87. Given that ts/tv averaged across the
+human genome is about 2 but ts/tv averaged over random errors is 0.5, the
+minimap2 callset arguably has higher precision at lower sensitivity.
+
+The sample being assembled is a female. Minimap2 still called 201 substitutions
+on the Y chromosome. These substitutions all come from one contig aligned at
+96.8\% sequence identity. The contig could be a segmental duplication
+absent from GRCh38. In constrast, dnadiff called 9070 substitutions on the Y
+chromosome across 73 SMRT contigs. This again implies our minimap2-based
+pipeline has higher precision.
+
+\section{Discussions}
+
+Minimap2 is a versatile mapper and pairwise aligner for nucleotide sequences.
+It works with short reads, assembly contigs and long noisy genomic and RNA-seq
+reads, and can be used as a read mapper, long-read overlapper or a full-genome
+aligner. Minimap2 is also accurate and efficient, often outperforming other
+domain-specific alignment tools in terms of both speed and accuracy.
+
+The capability of minimap2 comes from a fast base-level alignment algorithm and
+an accurate chaining algorithm. When aligning long query sequences, base-level
+alignment is often the performance bottleneck. The Suzuki-Kasahara algorithm
+greatly alleviates the bottleneck and enables DP-based splice alignment
+involving $>$100kb introns, which was impractically slow ten years ago.  The
+minimap2 chaining algorithm is fast and highly accurate by itself.  In fact,
+chaining alone is more accurate than all the other long-read mappers in
+Fig.~\ref{fig:eval}a (data not shown). This accuracy helps to reduce downstream
+base-level alignment of candidate chains, which is still several times slower than
+chaining even with the Suzuki-Kasahara improvement. In addition, taking a
+general form, minimap2 chaining can be adapted to non-typical data types such as
+spliced reads and multiple reads per fragment. This gives us the opportunity to
+extend the same base algorithm to a variety of use cases.
+
+Modern mainstream aligners often use a full-text index, such as suffix array or
+FM-index, to index reference sequences. An advantage of this approach is that
+we can use exact seeds of arbitrary lengths, which helps to increase seed
+uniqueness and reduce unsuccessful extensions. Minimap2 indexes reference
+k-mers with a hash table instead. Such fixed-length seeds are inferior to
+variable-length seeds in theory, but can be computed much more efficiently in
+practice. When a query sequence has multiple seed hits, we can afford to skip
+highly repetitive seeds without affecting the final accuracy. This further
+alleviates the concern with the seeding uniqueness. At the same time, at low
+sequence identity, it is rare to see long seeds anyway. Hash table is the ideal
+data structure for mapping long noisy sequences.
+
+\section*{Acknowledgements}
+We owe a debt of gratitude to H. Suzuki and M. Kasahara for releasing their
+masterpiece and insightful notes before formal publication. We thank M.
+Schatz, P. Rescheneder and F. Sedlazeck for pointing out the limitation of
+BWA-MEM. We are also grateful to minimap2 users who have greatly helped to
+suggest features and to fix various issues.
+
+\paragraph{Funding\textcolon} NHGRI 1R01HG010040-01
+
+\bibliography{minimap2}
+
+\end{document}