minimap2 0.2.23.0 → 0.2.23.1

data/ext/minimap2/tex/mm2-s3.sam.eval

@@ -0,0 +1,62 @@
+Q	60	18579866	27	0.000001453	18579866
+Q	59	27087	4	0.000001666	18606953
+Q	58	21435	1	0.000001718	18628388
+Q	57	45663	3	0.000001874	18674051
+Q	56	36031	2	0.000001978	18710082
+Q	55	18499	2	0.000002082	18728581
+Q	54	14754	2	0.000002187	18743335
+Q	53	25541	2	0.000002291	18768876
+Q	52	26397	5	0.000002554	18795273
+Q	51	15090	3	0.000002711	18810363
+Q	50	13425	11	0.000003294	18823788
+Q	49	15175	2	0.000003397	18838963
+Q	48	19407	4	0.000003606	18858370
+Q	47	11538	16	0.000004452	18869908
+Q	46	12558	17	0.000005349	18882466
+Q	45	40362	28	0.000006817	18922828
+Q	44	10465	13	0.000007500	18933293
+Q	43	10098	20	0.000008552	18943391
+Q	42	10682	19	0.000009549	18954073
+Q	41	9823	11	0.000010125	18963896
+Q	40	9685	16	0.000010963	18973581
+Q	39	10273	18	0.000011905	18983854
+Q	38	9515	18	0.000012847	18993369
+Q	37	9474	27	0.000014261	19002843
+Q	36	10430	25	0.000015568	19013273
+Q	35	9241	34	0.000017348	19022514
+Q	34	9162	31	0.000018968	19031676
+Q	33	10164	49	0.000021532	19041840
+Q	32	9152	55	0.000024408	19050992
+Q	31	9252	35	0.000026233	19060244
+Q	30	9872	55	0.000029103	19070116
+Q	29	8938	65	0.000032496	19079054
+Q	28	8951	73	0.000036306	19088005
+Q	27	9949	95	0.000041261	19097954
+Q	26	9784	97	0.000046316	19107738
+Q	25	10126	97	0.000051366	19117864
+Q	24	11260	123	0.000057765	19129124
+Q	23	10047	114	0.000063691	19139171
+Q	22	9661	123	0.000070083	19148832
+Q	21	10339	168	0.000078813	19159171
+Q	20	17928	193	0.000088804	19177099
+Q	19	9842	193	0.000098817	19186941
+Q	18	14737	247	0.000111605	19201678
+Q	17	10218	238	0.000123934	19211896
+Q	16	10271	242	0.000136457	19222167
+Q	15	12241	333	0.000153683	19234408
+Q	14	9189	336	0.000171070	19243597
+Q	13	9493	515	0.000197734	19253090
+Q	12	11502	743	0.000236185	19264592
+Q	11	8211	507	0.000262390	19272803
+Q	10	9133	606	0.000293695	19281936
+Q	9	10014	931	0.000341801	19291950
+Q	8	8436	698	0.000377816	19300386
+Q	7	8443	705	0.000414163	19308829
+Q	6	10203	944	0.000462808	19319032
+Q	5	6936	756	0.000501760	19325968
+Q	4	6732	843	0.000545190	19332700
+Q	3	8215	1104	0.000602040	19340915
+Q	2	21201	5440	0.000882342	19362116
+Q	1	82328	22186	0.002019600	19444444
+Q	0	553853	371953	0.020562901	19998297
+U	1703

data/ext/minimap2/tex/mm2-update.tex

@@ -0,0 +1,240 @@
+\documentclass{bioinfo}
+\copyrightyear{2021}
+\pubyear{2021}
+
+\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[Improvements to minimap2]{New strategies to improve minimap2 alignment accuracy}
+\author[Li]{Heng Li$^{1,2}$}
+\address{$^1$Dana-Farber Cancer Institute, 450 Brookline Ave, Boston, MA 02215, USA,
+$^2$Harvard Medical School, 10 Shattuck St, Boston, MA 02215, USA}
+
+\maketitle
+
+\begin{abstract}
+
+\section{Summary:} We present several recent improvements to minimap2, a
+versatile pairwise aligner for nucleotide sequences. Now minimap2 v2.22 can
+more accurately map long reads to highly repetitive regions and align through
+insertions or deletions up to 100kb by default, addressing major weakness in
+minimap2 v2.18 or earlier.
+
+\section{Availability and implementation:}
+\href{https://github.com/lh3/minimap2}{https://github.com/lh3/minimap2}
+
+\section{Contact:} hli@ds.dfci.harvard.edu
+\end{abstract}
+
+\section{Introduction}
+Minimap2~\citep{Li:2018ab} is widely used for maping long sequence
+reads and assembly contigs. \citet{Jain:2020aa} found minimap2 v2.18 or earlier occasionally
+misaligned reads from highly repetitive regions as minimap2 ignored seeds of
+high occurrence. They also noticed minimap2 may misplace reads with structural
+variations (SVs) in such regions~\citep{Jain2020.11.01.363887}. These
+misalignments have become a pressing issue in the advent of
+temolere-to-telomore human assembly~\citep{Miga:2020aa}. Meanwhile, old minimap2
+was unable to efficiently align long insertions/deletions (INDELs) and often
+breaks an alignment around variable-number tandem repeats (VNTRs). This has
+inspired new chaining algorithms~\citep{Li:2020aa,Ren:2021aa} which are not
+integrated into minimap2. Here we will describe recent efforts implemented
+in v2.19 through v2.22 to improve mapping results.
+
+\begin{methods}
+\section{Methods}
+
+\subsection{Rescuing high-occurrence $k$-mers}\label{sec:high-occ}
+Minimap2 keeps all $k$-mer minimizers~\citep{Roberts:2004fv} during indexing. Its original
+implementation only selected low-occurrence minimizers during mapping. The
+cutoff is a few hundred for mapping long reads against a human genome. If a
+read habors only a few or even no low-occurrence minimizers, it will fail
+chaining due to insufficient anchors.
+
+To resolve this issue, we implemented a new heuristic to add additional
+minimizers. Suppose we are looking at two adjacent low-occurence $k$-mers
+located at position $x_1$ and $x_2$, respectively. If $|x_1-x_2|\ge L$,
+minimap2 v2.22 additionally selects $\lfloor|x_1-x_2|/L\rfloor$ minimizers
+of the lowest occurrence among minimizers between $x_1$ and $x_2$. Here
+parameter $L$ controls the frequency of sampling. It defaults to 500.
+This strategy adds necessary anchors at the cost of increasing total alignment
+time by a few percent on real data.
+
+\subsection{Aligning through longer INDELs}
+The original minimap2 may fail to align long INDELs due to its chaining
+heuristics. Briefly, minimap2 applies dynamic programming (DP) to chain
+minimizer anchors. This is a quadratic algorithm, slow for chaining
+contigs. For acceptable performance, the original minimap2 uses a 500bp band by
+default, which means a gap longer than 500bp will stop chaining.
+To align through longer gaps, older minimap2 implemented a long-join heurstic as follows.
+If there is an INDEL longer than 500bp and the two chains around the INDEL
+have no overlaps on either the query or the reference sequence, minimap2 may
+join the two short chains later.
+This heuristic may fail around VNTRs because short chains
+often have overlaps in VNTRs. More subtly, minimap2 may escape the inner DP
+loop early, again for performance, if the chaining result is not improved for
+50 iterations. When there is a copy number change in a long segmental
+duplication, the early escape may break around the event even if users
+specify a large band.
+
+In minigraph~\citep{Li:2020aa}, we developed a new chaining algorithm that
+finds up to 1kb INDELs with DP-based chaining and goes through longer INDELs with a
+subquadratic algorithm~\citep{DBLP:conf/wabi/AbouelhodaO03}. We ported the same
+algorithm to minimap2 for contig mapping. For long-read mapping, the minigraph
+algorithm is slower. Minimap2 v2.22 still uses the DP-based algorithm to
+find short chains and then invokes the minigraph algorithm to rechain anchors in
+these short chains. The rechaining step achieves the same goal as long-join
+but is more reliable because it can resolve overlaps between short chains. The old
+long-join heuristic has since been removed.
+
+\subsection{Properly mapping long reads with SVs}
+The original minimap2 ranks an alignment by its Smith-Waterman score and
+outputs the best scoring alignment. However, when there are SVs on the read,
+the best scoring alignment is sometimes not the correct alignment.
+\citet{Jain2020.11.01.363887} resolved this dilemma by altering the mapping
+algorithm.
+
+In our view, this problem is rooted in inapropriate scoring: affine-gap penalty
+over-penalizes a long INDEL that was often evolutionarily created in one event.
+We should not penalize a SV by a function linear in the SV length. Minimap2 v2.22 instead rescores
+an alignment with the following scoring function. Suppose an alignment consists
+of $M$ matching bases, $N$ substitutions and $G$ gap opens, we empirically
+score the alignment with
+$$
+S=M-\frac{N+G}{2d}-\sum_{i=1}^G\log_2(1+g_i)
+$$
+where $g_i\ge1$ is the length of the $i$-th gap and
+$$
+d=\max\left\{\frac{N+G}{M+N+G},0.02\right\}
+$$
+It approximates per-base sequence divergence except with the smallest value set
+to 2\%. As an analogy to affine-gap scoring, the matching score in our scheme
+is 1, the mismatch and gap open penalties are both $1/2d$ and the gap extension
+penalty is a logarithm function of the gap length~\citep{Gu:1995wt}. Our scoring gives a long SV
+a much milder penalty. In terms of time complexity, scoring an alignment is
+linear in the length of the alignment. The time spent on rescoring is negligible in
+practice.
+
+%If we assume sequences evolve under a duplication-mutation model, we may have a
+%better way to choose the best alignment. If a long read can be mapped to $n$
+%loci, we can take the read as the template and build a
+%pseudo-multi-sequence-alignment (pMSA) of $n+1$ sequences. In this pMSA, we say
+%a site on the read is informative if the $n$ reference subsequences differ at
+%the position.
+
+\end{methods}
+
+\section{Results}
+
+\begin{table}
+\processtable{Evaluation of minimap2 v2.22}
+{\footnotesize\label{tab:1}\begin{tabular}{p{4.2cm}rrrr}
+\toprule
+$[$Benchmark$]$ Metric & v2.22 & v2.18 & Winno & lra \\
+\midrule
+$[$sim-map$]$ \% mapped reads at Q10      & 97.9       & 97.6      & {\bf 99.0}& 97.3 \\
+$[$sim-map$]$ err. rate at Q10 (phredQ)   & {\bf 52}   & {\bf 52}  & 38        & 24 \\
+$[$winno-cmp$]$ rate of diff. (phredQ)    & {\bf 41}   & 37        & truth     & 18 \\
+$[$winno-cmp$]$ CPU time (hour)           & {\bf 5.0}  & 5.3       & 71.8      & 13.1 \\
+$[$winno-cmp$]$ peak RAM (Gb)             & 17.1       & 14.4      & {\bf 9.6} & 12.4 \\
+$[$sim-sv$]$  \% false negative rate      & {\bf 0.5}  & 2.0       & {\bf 0.5} & 1.4  \\
+$[$sim-sv$]$  \% false discovery rate     & {\bf 0.0}  & 0.1       & {\bf 0.0} & 0.1  \\
+$[$real-sv-1k$]$ \% false negative rate   & {\bf 7.3}  & 20.0      & 13.0      & N/A \\
+$[$real-sv-1k$]$ \% false discovery rate  & 2.7        & {\bf 2.4} & 2.7       & N/A \\
+\botrule
+\end{tabular}}
+{In $[$sim-map$]$, 152,713 reads were simulated from the CHM13 telomere-to-telomere assembly v1.1
+(AC: GCA\_009914755.3) with pbsim2~\citep{Ono:2021aa}: ``pbsim2 -{}-hmm\_model R94.model -{}-length-min
+5000 -{}-length-mean 20000 -{}-accuracy-mean 0.95''. Alignments of mapping quality
+10 or higher were evaluated by ``paftools.js mapeval''. The mapping error rate
+is measured in the phred scale: if the error rate is $e$, $-10\log_{10}e$ is
+reported in the table. In $[$winno-cmp$]$, 1.39 million CHM13 HiFi reads from
+SRR11292121 were mapped against the same CHM13 assembly. 99.3\% of them were mapped by Winnowmap2
+at mapping quality 10 or higher and were taken as ground truth to evaluate
+minimap2 and lra with ``paftools.js pafcmp''. $[$sim-sv$]$ simulated 1,000
+50bp to 1000bp INDELs from chr8 in CHM13 using SURVIVOR~\citep{Jeffares:2017aa} and simulated Nanopore
+reads at 30-fold coverage with the same pbsim2 command line. SVs were called with
+``sniffles -q 10''~\citep{Sedlazeck:2018ab} and compared to the simulated truth with ``SURVIVOR eval
+call.vcf truth.bed 50''. In $[$real-sv-1k$]$, small and long variants were
+called by dipcall-0.3~\citep{Li:2018aa} for HG002 assemblies (AC: GCA\_018852605.1 and
+GCA\_018852615.1) and compared to the GIAB truth~\citep{Zook:2020aa} using ``truvari -r 2000 -s
+1000 -S 400 -{}-multimatch -{}-passonly'' which sets the minimum INDEL size to 1kb in evaluation. }
+\end{table}
+
+We evaluated minimap2 v2.22 along with v2.18, Winnowmap2 v2.03 and lra v1.3.2
+(Table~\ref{tab:1}), using the default setting of each mapper according to the input data types.
+Both versions of minimap2 achieved high mapping accuracy on
+simulated Nanopore reads (sim-map). Winnowmap2 aligned more reads at mapping
+quality 10 or higher (mapQ10). However, it may occasionally assign a high mapping
+quality to a read with multiple identical best alignments. This reduced its
+mapping accuracy.
+
+In lack of groud truth for real data, we took Winnowmap2 mapping as ground
+truth to evaluate other mappers (winno-cmp in Table~\ref{tab:1}). Out of 1,378,092 reads with mapQ10
+alignments by Winnowmap2, minimap2 v2.22 could map all of them. 118 reads, less
+than 0.01\% of all reads, were mapped differently by v2.22. 51 of them have
+multiple identical best alignments. We believe these are more likely to be
+Winnowmap2 errors. Most of the remaining 67 (=118-51) reads have multiple
+highly similar but not identical alignments.
+Minimap2 v2.18 is less consistent with 275 differences including 30 unmapped
+reads mappable by both Winnowmap2 and v2.22.
+
+For the minimizer rescuing parameter $L$ in Section~\ref{sec:high-occ},
+we set its default to 500 such that v2.22 has comparable performance to v2.18 given simulated PacBio and Nanopore human reads.
+To see the effect of this parameter on real data, we tried several different $L$ values.
+v2.22 gave 99 mapping differences at $L=200$,
+118 at $L=500$ (default), 167 at $L=750$ and 224 differences at $L=1000$ in comparison to Winnowmap2.
+$L=200$ is 28\% slower than the default while $L=1000$ is 9\% faster.
+Changing the default minimizer window size (option ``-w'')
+and the initial minimizer occurrence cutoff (option ``-f'')
+also affects performance and accuracy to a similar magnitude.
+
+The two benchmarks above only evaluate read mappings when there are no variations between the reads and the reference.
+To measure the mapping accuracy in the presence of SVs (sim-sv), we reproduced
+the results by~\citep{Jain2020.11.01.363887}. Minimap2 v2.22 is as good as
+Winnowmap2 now. Note that we were setting the Sniffles mapping quality
+threshold to 10 in consistent with the benchmarks above. If we used the
+default threshold 20, v2.22 would miss additional five SVs (accounting for
+0.5\% of simulated SVs). For four out of these five missing SVs, minimap2 v2.22
+mapped more variant reads than Winnowmap2. Sniffles did not call these SVs
+because minimap2 tended to give them conservative mapping quality. It is worth
+noting that the simulation here only considers a simple scenario in evolution.
+Non-allelic gene conversions, which happen often in segmental
+duplications~\citep{Harpak:2017aa}, would obscure the optimal mapping
+strategies. How much such simple SV simulation informs real-world SV calling
+remains a question.
+
+To see if minimap2 v2.22 could improve long INDEL alignment, we ran dipcall on
+contig-to-reference alignments and focused on INDELs longer than 1kb
+(real-sv-1k). v2.22 is more sensitive at comparable specificity, confirming its
+advantage in more contiguous alignment. We could not get dipcall to work well with lra,
+so did not report the numbers.
+
+Minimap2 spends most computing time on base alignment. As recent improvements
+in v2.22 incur little additional computing and do not change the base alignment
+algorithm, the new version has similar performance to older versions. It is
+consistently faster than Winnowmap2 by several times. Sometimes simple
+heuristics can be as effective as more sophisticated yet slower solutions.
+
+\section*{Acknowledgements}
+We thank Arang Rhie and Chirag Jain for providing motivating examples for which
+older minimap2 underperforms.
+
+\paragraph{Funding\textcolon} This work is funded by NHGRI grant R01HG010040.
+
+\bibliography{minimap2}
+
+\end{document}

data/ext/minimap2/tex/mm2.approx.eval

@@ -0,0 +1,12 @@
+Q	60	32084	0	0.000000000	32084
+Q	24	318	2	0.000061725	32402
+Q	11	98	2	0.000123077	32500
+Q	8	37	2	0.000184405	32537
+Q	7	37	3	0.000276294	32574
+Q	6	40	3	0.000367940	32614
+Q	5	34	2	0.000428816	32648
+Q	4	37	5	0.000581306	32685
+Q	3	28	6	0.000764222	32713
+Q	2	38	6	0.000946536	32751
+Q	1	50	21	0.001585318	32801
+Q	0	286	150	0.006105117	33087

data/ext/minimap2/tex/mm2.eval

@@ -0,0 +1,13 @@
+Q	60	32477	0	0.000000000	32477
+Q	22	16	1	0.000030776	32493
+Q	21	44	1	0.000061468	32537
+Q	19	73	1	0.000091996	32610
+Q	14	66	1	0.000122414	32676
+Q	10	26	3	0.000214054	32702
+Q	8	14	1	0.000244529	32716
+Q	7	13	2	0.000305539	32729
+Q	6	47	1	0.000335611	32776
+Q	3	10	1	0.000366010	32786
+Q	2	20	2	0.000426751	32806
+Q	1	248	94	0.003267381	33054
+Q	0	31	17	0.003778147	33085