# Supporting R9 data in nanopolish

Two weeks ago I mentioned on twitter that I pushed a large series of changes to support R9 Oxford Nanopore data in nanopolish. I promised a blog post describing the changes then went off on my summer vacation - here it is, a little late.

First, some background about R9 data. Back in March Clive Brown held a google hangout to announce that ONT was switching to a new pore. The old pore, dubbed R7, would be discontinued and a new pore based on the CsgG lipoprotein, called R9, would take its place. A host of other changes came with the pore swap; the sequencing speed would be increased (from 70bp/s to 250bp/s) and the ONT basecaller would now use a recurrent neural network rather than a hidden Markov model. Supporting this data would require quite a few changes to nanopolish - we would need a new pore model (a set of parameters for the Gaussian emission distributions in our HMM), new transition probabilities for the model (due to the increased speed) and we could no longer rely on extracing the parameters that scale a read to the pore model from the FAST5 files as the RNN does not calculate these.

The R9 pore model was the simplest issue to resolve - ONT provided a new table mapping k-mers to Gaussian parameters. We then wrote code to infer the per-read scaling parameters from the output of the RNN, which assigns 5-mer labels to events. Nick and Josh generated some early R9 data and I worked on updating the new transition parameters in the HMM. The initial polishing experiments went ok - we got up to about 99.7% accuracy - but fell short of our best R7 accuracy, which was about 99.8%. After I returned from London Calling I spent a painful few weeks looking at regions of the E. coli genome that were miscalled. I eventually realized that the change in sequencing speed had a bigger effect than I realized - rather than just changing the transition parameters of the HMM I would have to change the structure as well. The key issue is that R9 events are oversegmented (there are more events than base movements through the pore) rather than undersegmented, like in R7. The more aggressive segmentation makes the events more susceptible to transient noise causing their level to be drastically different than the model. To fix this issue we added a new state to the HMM which would allow it to completely ignore an event. The transitions to this state have very low probability to avoid allowing the HMM to ignore many events.

In the midst of all of these model changes I rewrote the consensus algorithm. The original consensus algorithm had a rather clunky system for keeping track of where the reads aligned to the assembly. This system made development and debugging tedious so I merged the consensus functionality with the much better code that we wrote to call SNPs for Ebola. The README on github contains instructions for using the new consensus workflow.

## R9 Polishing Results

ONT provided my lab with a batch of the latest flowcells. Phil Zuzarte broke our lab record for throughput by generating almost 1Gbp of 1D reads and 450 Mbp of 2D reads for E. coli K12. We used the latest version of canu to generate a draft assembly of this data and computed a new consensus using the updated nanopolish. As before we evaluated our assembly using nucmer which reported 99.96% identity to the K12 reference. This is a remarkable improvement from our first polishing run from January 2015, which only reached 99.48% identity. As an indication of just how much the data has improved in the last year, the unpolished canu assembly from this run has better accuracy (99.66%) than that first polished assembly.

Clive suggested we try to mix R7 and R9 data to jointly call a consensus, which bumped identity up to 99.98%. This hints that mixing data with different signal properties - might be a direction to explore.

## Homopolymers

These results are encouraging but we want to improve accuracy further. Most of the remaining errors are (predictably) in homopolymer runs. Interestingly we are able to call some longer homopolymers (7,8,9 bp runs) correctly now using the new HMM (which does not penalize homopolymers so if there are multiple events for a homopolymer they will not be collapsed to a single state). However accuracy for long homopolymers remains the biggest problem. To address this we have started to explore explicitly modeling the duration of events. This should be a major focus in the upcoming months.

So far we have mostly tested the new algorithm on E. coli data - if anyone else has a high-depth R9 data set and would like to give the new code a try we would love to hear from you.

Thanks to Matei David, Jonathan Dursi, Nick Loman, Josh Quick and Phil Zuzarte for helping develop the R9 support and generating data. The scientists at Oxford Nanopore helped us better understand R9 data and the discussions we had with them were key to implementing the improved HMM - thanks in particular to Clive and Chris Wright.

Disclosure: Oxford Nanopore provides research funding to my lab.

# Deprecating Nanocorrect

When Nick, Josh and I got together at the Newton Institute hackathon in 2014 we wanted to see if assembling nanopore data was possible. Over the week we put together a pipeline inspired by pbdagcon which used DALIGNER and poa to correct sequencing errors in nanopore reads. This software, which we called nanocorrect, improved the accuracy of our nanopore reads to around 97% after two rounds of correction. The corrected reads worked well in the Celera Assembler and we had very long contigs soon after the hackathon.

To complete our de novo assembly paper we wrote a second, much more powerful, software package called nanopolish that uses the nanopore signal data to improve the accuracy of the assembly. Nanopolish has since grown to support aligning signal events to a reference genome and calling SNPs. Where nanocorrect was a simple, quick solution to error correction, nanopolish is a long term project that I’ve spent most of my time on in the last year.

Nanocorrect’s major flaw is that it is very slow (specifically the poa step which constructs a partial order alignment between sets of overlapping reads). In the year since we published our paper a few other nanopore-compatible assemblers have appeared, namely canu and miniasm. These programs are clearly better at building contigs than our nanocorrect + CA pipeline so we’ve decided to deprecate nanocorrect. Our current suggestion is to build contigs with canu and use nanopolish to compute the final consensus sequence.

# Finding Ebola SNPs

Today Nature published our paper describing the use of the Oxford Nanopore MinION to track the Ebola outbreak in West Africa. Over on Nick Loman’s blog you can read an excellent account of how this project started and progressed. For my part, I worked on the method that we used to call SNPs from the sequenced Ebola samples, which I’ll describe in this post.

## Background

Nanopore sequencing measures the changes in electric current (termed events) caused by DNA translocating through the pore. Previously I developed a hidden Markov model for calculating the probability of observing a particular sequence of events given a DNA sequence. We’ve described this HMM in our genome assembly paper and in previous blog posts here and here. I won’t describe the details again but the important thing to understand for this post is that we have a function $P(D \; \vert \; S)$ that calculates the probability of observing some data $D$ (measured by the nanopore sequencer) given a sequence $S$ (that we want to determine). In our genome assembly paper we used the HMM to optimize a consensus sequence by iteratively editing $S$ until we couldn’t make any further improvements.

## The first algorithm

Last summer Nick and Matt Loose organized a hackathon in Birmingham where I started to work on the SNP calling problem. I thought this would be straightforward as SNP calling is a special case of calculating a consensus sequence. If SNPs are well-separated from each other we can simply calculate the likelihood of a haplotype containing a SNP and compare it to the likelihood of the reference haplotype:

$P(D \; \vert \; \texttt{...ACGATCGTACA...})$ (reference)
$P(D \; \vert \; \texttt{...ACGATAGTACA...})$ (snp haplotype)

If $P(D \; \vert \; \texttt{...ACGATAGTACA...}) > P(D \; \vert \; \texttt{...ACGATCGTACA...})$ we would call a C to A SNP and output it in a VCF file with a corresponding quality score. This simple approach worked well for most SNPs in our test case. We observed however that we would miscall regions that had multiple nearby SNPs. Recall from the previous posts that the nanopore sequencer does not measure signals from individual bases; it effectively reads k-mers, not bases. When trying to call nearby SNPs unless we present the complete true haplotype sequence to the algorithm it may either only find a subset of the true SNPs or worse, make false calls. Clearly we had to jointly test SNPs in groups rather than individually.

## The failed algorithm

I spent the remainder of the hackathon working on an idea that would more generally solve the consensus problem. Let’s say we know the true sequence of some partial region of the genome, $S’$. Can we determine the base immediately following $S'$? On the surface this is simple: we can select the extension with the greatest likelihood out of the four possibilities:

$P(D \; \vert \; \texttt{S'A})$
$P(D \; \vert \; \texttt{S'C})$
$P(D \; \vert \; \texttt{S'G})$
$P(D \; \vert \; \texttt{S'T})$

To do this calculation we need to modify our HMM. Previously we required the event sequence to be completely aligned - we knew the first and last event observation for the region of interest. For this problem, we need to relax this constraint and allow uncertainty about the endpoint of the sequence of events. We modified our HMM to handle this by allowing unmatched events at the beginning and end of the matching region, similar to how bases may be soft-clipped in nucleotide alignments.

With the HMM modified we can calculate likelihoods for the one-base extensions of S’. By iterating the procedure we would hopefully arrive at the true sequence of the region including any variants. Selecting the single best extension at each step worked extremely poorly. If an error was made (the wrong extension was selected) the error would propagate and we would have a nonsense reconstruction of the region. I tried various heuristics for keeping track of the N best solutions to avoid forcing the algorithm to take a single choice. This can be incredibly expensive however and I did not find a solution that worked well in all cases. When this algorithm goes wrong we would get a dense cluster of false positive SNPs, which is not a good property of an algorithm. I abandoned this approach shortly after I left the hackathon.

## The final algorithm

The iterative algorithm which performed reasonably well, but not perfectly, for variant calling ignores an additional source of information: the basecalled sequences of the reads. We decided to find candidate SNPs from the nanopore reads aligned to the reference genome and test combinations of these candidates. We batch SNPs into clusters that are separated by about 10bp, a value I determined that allows the clusters to be treated independently. We then simply generate all possible haplotypes ($2^n$ for $n$ SNPs) and calculate the likelihood for each haplotype using the HMM. The haplotype with the greatest likelihood is selected as the sequence for the region and its set of SNPs is output in the VCF file.

I sent this version of the algorithm to Nick who tested it on the Mayinga strain of Ebola which has ~500 mutations when compared to the Makona reference (in 19,000 bases, i.e. >2.6% divergence - samples from the West Africa outbreak typically have 20-40 SNPs and are much easier to call). This haplotype-based version was far better than the previous algorithms and forms the basis of the method we used in the paper.

We made a final improvement which used a new 6-mer pore model from Oxford Nanopore. To use this model on our data, which was sequenced with the older SQK005 sequencing kit (and basecalled using 5-mers), we had to calculate a new scaling parameter to transform the events to the 6-mer model. Using 6-mers was more accurate in difficult sequence contexts and completed the algorithm development. The calls from this algorithm are presented in the paper. The SNP caller is implemented in the new “variants” module of nanopolish.

# Approximate Mapping of Nanopore Squiggle Data with Spatial Indexing

### Nanopore data

Here at the Simpsonlab we work with signal-level data from nanopore sequencers, particularly those from ONT like the MinION. Signal-level data looks something like this:

where the data comes in as a stream of events (the red lines) with means, standard deviations, and durations, with each black point being an individual signal reading. We use the signal-level data partly for accuracy - because converting each red line and dozens of black points into one of four letters invariably loses lots of information - and partly because those basecalls might not be available in, say, the field, where the portability of the nanopore sequencers really shines.

This data has to be interpreted in terms of a particular model of the interaction of the pore and the strand of DNA; such a pore model gives, amongst other things, an expected signal mean and standard deviation for currents through the pore when a given $k$mer is in the pore (for the MinION devices, pore models typically use $k$=5 or 6). So a pore model might be, for $k$=5, a table with 1024 entries that looks something like this:

kmer mean std dev
AAAAA 70.24 ± 0.95 pA
AAAAC 66.13 ± 0.75 pA
AAAAG 70.23 ± 0.76 pA
AAAAT 69.25 ± 0.68 pA

and using such a pore model it’s fairly straightforward to go from a sequence to an expected set of signal levels from the sequencer: so, say, for the 10-base sequence AAAAACGTCC and the above 5-mer pore model we’d expect 6 events (10-5+1) that look like:

event kmer mean range (1$\sigma$)
e1 AAAAA 70.2 pA 69.3 - 71.2 pA
e2 AAAAC 66.1 pA 65.4 - 66.9 pA
e3 AAACG 62.8 pA 62.0 - 63.6 pA
e4 AACGT 67.6 pA 66.3 - 68.9 pA
e5 ACGTC 56.6 pA 54.5 - 58.7 pA
e6 CGTCC 49.9 pA 49.1 - 50.7 pA

Using those models, plus HMMs and a tonne of math, Jared’s nanopolish tool has had enormous success improving the quality of nanopore reads and assemblies of nanopore data. As one can see from the ‘range’ column above, and from the histogram on the side of the first plot, which shows the numbers of kmers which fall into 1pA bins in the pore model, this isn’t necessarily particularly easy. There are over 70 kmers which are expected to have means in the range 69.5pA - 70.5pA; that plus the wide standard deviations means that there is enormous ambiguity going back from signal levels to sequence.

### Mapping

This ambiguity introduces complication into another part of the bioinformatics pipeline - mapping. While several mappers (BWA MEM, LAST) work quite well with basecalled ONT data, and one mapper1 has been written specifically for basecalled ONT data, mapping signal-level data directly is a little tougher. It is one thing for a seed sequence in the read to occur in multiple locations in the reference index; it is another thing for a sequence of floating-point currents to plausibly represent one of hundreds of seed sequences.

On the other hand, a flurry of recent important papers and software2 3 4 5, the first of which are discussed in some informative blog posts, have demonstrated the usefulness of approximate read mapping. In our case, mapping to an approximate region of a reference would allow exact but computationally expensive methods like nanopolish eventalign to produce precise alignments, or simple assignment may be all that is necessary for other applications.

So an output approximate mapping would be valuable, but the issues remains of how to disambiguate the signal level values.

### Spatial Indexing

Importantly, while any individual event is very difficult to assign with precision, sequences of events are less ambiguous, as any given event is followed, with high probability, by one of only four other events. Thus, by examining tuples of events, one can greatly increase specificity.

And there are well-developed sets of techniques for looking up lists of $d$ floating point values to look for candidate matches: spatial indexes, where each query or match is considered as a point in $d$-dimensional space, and the goal is to return either some number of nearest points (k-Nearest-Neighbours, or kNN queries), or all matches within some, typically euclidean, distance.

To see how this would work, consider indexing the sequence above, AAAAACGTCC, in a spatial index with $d=2$. There are a total of 6 events, so we’d have 5 points (6-2+1), each points in 2-dimensional space; 4 are plotted below (the other falls out of the range of the plot)

and a query for $d$ read events that could be matched by (69.5, 67), the blue point, would return the nearest match (70.2, 66.1) corresponding to the $k+d-1$-mer AAAAAC.

So to map event data, one can:

• Generate an index:
• Generate all overlapping tuples of $d$ kmers from a reference
• Using a pore model, convert those into points in $d$-space
• Create a spatial index of those points,
• For every read:
• Take every overlapping set of $d$ events
• Look it up in a spatial index
• Find a most likely starting point for the read in the reference, with a quality score.

Note that one has to explicitly index both the forward and reverse strands of the reference, since you don’t a priori know what the “reverse complement” of 65.5 pA is. One also has to generate multiple indices; for 2D ONT reads, there is a pore model for the template strand of a read, and typically two possible models to choose from for the complement strand of a read, so one needs three indexes in total to query.

### What dimension to choose?

To test this approach, you have to choose a $d$ to use. On the one hand, you would like to choose as large a dimension as you can; the larger $k+d-1$ is, the more unique each seed is and the fewer places it will occur in any reference sequence.

On the other hand, two quite different constraints put a strong upper limit on the dimensions that will be useful:

• The curse of dimensionality - in high spatial dimensions, nearest-neighbour searching is extremely inefficient, because distance loses its discriminative power. (Most things are roughly equally far from each other in 100-dimensional space; there are a lot of routes you can take!) As a practical matter, for most spatial index implementations, for $d > 15-20$ or so you might as well just do a linear search over all possible items;
• Current approaches to segmentation mean that ONT data has very frequent ‘stops’ and ‘skips’ - that is, events are spuriously either inserted or deleted from the data. Exactly as with noisy basecalled sequences, this strongly limits the length of sequence that can be usefully used as seeds. As we see below for one set of E. coli data, there is probably not much point in using $d \ge 10$ even for template-strand data, as the median “dmer” will have an insertion/deletion in it.

For these two reasons, we’ve been playing with $d \approx 8$. I’ll note that while increasing $d$ is the most obvious knob to turn to increase specificity of the match, higher $k$ helps as well.

### Normalizing the signal levels

One issue we haven’t mentioned is that the raw nanopore data needs calibration to be compared to the model; there is an additive shift and drift over time, and a multiplicative scale that has to be taken into account.

A very simple “methods of moments” calculation works surprisingly well for long-enough reads, certainly well enough to start an iterative process; for any given model one is trying to fit a read to, rescaling the mean and standard deviation of read events to model events gives a good starting point for calibration, and drift is initially ignored.

### Proof of concept

A simple proof of concept of using spatial indexing to approximately map squiggle data can be found on github. It’s written in python, and has scipy, h5py, and matplotlib as dependencies. Note that as implemented, it is absurdly memory-hungry, and definitely requires a numpy and scipy built against a good BLAS implementation.

As a spatial index, it uses a version of a k-d tree (scipy.spatial.cKDTree), which is a very versatile and widely used (and so well-optimized) spatial index widely used in machine learning methods amongst others; different structures may have advantages for this application.

Running the index-and-map.sh script generates an index for the provided ecoli.fa reference - about 1 minute per pore model - and then maps the 10 reads provided of both older 5mer and newer 6mer MAP data. Mapping each read takes about 6 seconds per read per pore model; this involves lots of python list manipulations so could fairly straightforwardly be made much faster.
Doing the simplest thing possible for mapping works surprisingly well. Using the same sort of approach as the first steps of the Sovic et al. method1, we just:

• Use the default k-d tree parameters (which almost certainly isn’t right, particularly the distance measure)
• Consider overlapping bins of starting positions on the reference, of size ~10,000 events, a typical read size
• For each $d$-point in the read,
• Take the closest match to each $d$-point (or all within some distance)
• For each match, add a score to the bin corresponding to the implied starting position of the read on the reference; a higher score for a closer match
• Report the best match starting point

Let’s take a look at the initial output for the older SQK005 ecoli data and newer SQK006 data:

We see a couple of things here:

• Adding the complement strand does almost nothing for the accuracy, but requires substantially more memory and compute time, as multiple indices must be loaded and used up, and all candidate complement indices must be compared against each other. Because of this and the typically higher skip/stay rates for complement strands, we will use the template strand only for the rest of this post;
• Since we are simply assigning starting bins at this point, any assignments within the bin size are equally accurate; here, all of the reads were correctly assigned to the starting bin or the neighbouring one.
• The newer 6mer data gives slightly better results; part of this is likely because we are using the same $d$, so a dmer for the k=6 data corresponds to seed longer by one
• The zscore here is a very crude measure of how much the assignment stands out over the background (but not necessarily how it compares to other candidate mappings); some of these very simple pseudo-mappings are relatively securely identified, and others less so. The sum-of-scores for the reads with the best and worst zscore results are plotted below; no prize for guessing which is which:
 5mer 6mer

Because many levels are clustered near ~60-70pA, many dmers are quite close to each other, and choosing simply the closest $d$-point is unlikely to give a robust result. Examining all possible matches in the spatial index within some given radius reduces the noise somewhat, at a modest increase in compute time:

Note the increase in zscores; the same two reads are plotted:

 5mer 6mer

With a few other tweaks - keeping track of the top 10 candidates, and for each re-testing by recalibrating given the inferred mapping and rescoring - we can get about 95%-99% accuracy in mapping.

Of course, while 95-99% (Q13-Q20) mapping accuracy on E. coli is a cute outcome from such a simple approach, it isn’t nearly enough; with $d=8$ and $k=5$, wer’e working with seeds of size 12, which would typically be unique in the E. coli reference, but certainly wouldn’t be in the human genome, or for metagenomic applications.

### EM Rescaling

One limiting factor is the approximate nature of the rescaling so far that is being performed; for reads that have been basecalled, the inferred shift values above can be off by several picoamps from the Metrichor values, which clearly causes both false negatives and false positives in the index lookup. This can be addressed by doing a more careful rescaling step, using EM iterations:

• For the E-step, provisionally assign probabilities of read events corresponding to model levels, based on the Gaussian distributions described in the model and a simple transition matrix between events;
• For the M-step, perform a weighted least squares regression to re-scale the read levels.

This gives answers that are quite good when compared with Metrichor, at the cost of substantially more computational effort (much greater than the spatial index lookup!), particularly for long reads and larger (k) where the number of model levels is larger:

Note again the increase in zscores; replotting gives:

 5mer 6mer

### Extending the seeds

So far we have in no way taken into account any of the locality information in the spatial index lookup results; that is, that a long series of hits close together, in the same order on the read and on the reference, is much stronger evidence for a good mapping than a haphazard series of hits in random order that happen to fall within the same bin of starting points.

Keeping with the do-the-simplest-thing approach that has worked so far, we can try to extend these “seed” matches by stitching them together into longer seeds; here we build 15-mers out of sets of 4 neighbouring 12-mers, allowing one skip or stay somewhere within them, using as a score for the result the minimum of the constituent scores, and dropping all hits that cannot be so extended. This has quite modest additional cost, and works quite well:

 5mer 6mer

Now that we seem to have much stronger and more specific results, we can investigate letting go of binning, and instead string these extended seeds into longest possible matches. Continuing to extend dmer-by-dmer is too challenging due to missing points (due to mis-calibration, too large noise in the dmer falling out of the maxdist window in the kdtree, or several skip/stays in a row), so following the ideas of graphmap1 and minimap5, we trace collinear seeds through the set of available seeds; rather than just taking longest increasing sequences in inferred start positions, however, we make a graph of seeds with edges connecting seeds that are monotonically increasing both in read location and reference location with ‘small enough’ jumps, and extract longest paths through this graph. The cost of this is smaller than the rescaling of the read.

Running this, the zscores now somewhat change meaning; only the extracted paths are scored, meaning that the scores are only amongst plausible mappings, not over all bins across the reference. Similarly, the differences in mapping locations are somewhat more meaningful - rather than being compared to bin centres, they are actually the differences between mapping locations.

We get:

 5mer 6mer

The simple python testbed implementation of these ideas linked to above is very slow, single-threaded, absurdly memory hungry, and its treatment of scores for the mappings does not currently make much sense. In the new year we will address these issues in a proper C++ implementation, where (for instance) we will not have multiple copies of large, reference-sized data structures.

# Nanopolish v0.4.0

This is a long overdue post describing recent changes to nanopolish.

### SQK006 Support

Last month Oxford Nanopore released a new sequencing kit, SQK006. As Nick notes there are two important changes that affect how we model the data. Most importantly, ONT changed their basecaller to use 6-mers rather than 5-mers. The relationship between the DNA sequence in the pore and the measured signals is not simple (to understate the problem). By moving to a signal model with longer context the effect of long-range interactions that are not captured by shorter k-mer is hopefully reduced, which may improve read accuracy. In nanopolish v0.4.0 we now support the 6-mer model. This required reworking our data structures to allow either a 5-mer or 6-mer model depending on what sequencing kit was used to generate the data.

SQK006 also brings an increase in speed - from 30bp/s (SQK005) to 70bp/s (SQK006). With the sampling rate fixed to 3kHz we naturally expect more events to be missed by the event detector. We observed this in our analysis of SQK006 data and updated the initial transition probabilities in our HMM accordingly.

#### SQK006 Accuracy

We were quite curious to see how well the new data performs in practice. Nick and Josh made four runs with SQK006 kits - two of native E. coli DNA and two of PCR-treated E. coli DNA to remove DNA damage, base modifications and other artefacts. I downloaded one of the native runs and one of the PCR-treated runs and used it to make a new consensus sequence for the draft assembly in our paper. As before we use dnadiff from mummer to calculate percent identity, the number of SNPs and the number of indels with respect to the E. coli K12 reference genome.

Kit, Coverage Percent Identity # SNPs # Indels
SQK005, 29X 99.48% 1,343 22,601
SQK006, 48X 99.78% 644 9,697
SQK006-PCR, 30X 99.82% 222 8,200

The accuracy of our assembly is much better with SQK006 data. Most of this improvement is due to better representation of homopolymer sequences. Interestingly the PCR-treated run gave the best results despite being lower coverage than the native DNA run. It is tempting to attribute this to providing cleaner DNA to the nanopore but this requires further exploration first.

### Eventalign

In a previous post I introduced the eventalign submodule, which uses the hidden Markov model to align events to a reference genome. In nanopolish v0.4.0 there are a few eventalign changes and new features. The bulky model_name field has been removed from the output - it bloated the file as every row for a particular read would contain the same information. This read-level information has been moved to a secondary output file which can be enabled with the --summary FILE option. The summary file contains the path to the FAST5 file and model name for each read, along with the number of aligned events and other metadata that may be useful for exploring the alignments.

#### Experimental SAM output

eventalign now has an optional --sam flag which will write the alignment in a modified version of the SAM format. By example:

example_2d_read.template    0   chr1    10001   60  9950S4M1I1M2I1M1... *   0   0   *   * ES:i:1


Most of these fields should be familiar - the alignment contains a read name (annotated with the sequenced strand) followed by flags, a reference name and the alignment start position. Similar to a base-to-base alignment the CIGAR string describes the alignment of events to reference k-mers. 9950S4M indicates 9950 events should be skipped. The next event, 9951, aligns to the k-mer starting at reference position 10001, followed by three consecutive matches and so on. Sequence and quality information is not stored. The ES auxiliary tag is the event stride - whether the event indices are increasing along the reference sequence (ES:i:1) or whether they are decreasing (ES:i:-1). The nanopolish parser for this format is here.

There are a few reasons for using this format. First, it is much smaller than the eventalign tsv output. Second, by adopting sam/bam instead of creating a new binary format we can use the existing samtools/htslib ecosystem. Crucially, we can use htslib to provide random access into the alignments for an indexed bam file. This format was developed through discussions with Vadim Zalunin, Ewan Birney, Mick Watson and others. Comments welcome.