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12

I attended a talk by Josh Quick at PoreCampAU 2017, in which he discussed some common barriers to getting both good sequencing yield and long read length. It mostly boils down to being more careful with the sample preparation. Bear in mind that the MinION will still sequence a dirty sample, it will just be at a reduced yield. Here are my notes from that talk:...


8

Short answer: yes, but you need to get permission (and modified software) from ONT before doing that. ... but that doesn't tell the whole story. This question has the potential to be very confusing, and that's through no fault of the questioner. The issue is that for the MinION, sequencing (or more specifically, generating the raw data in the form of an ...


8

The quick way to get the number of alignments on each reference is samtools idxstats my_bam.bam Number of reads on each reference is column 3. Although, as has been pointed out, this will give you the total number of alignments per reference, not the total number of reads (each read might give rise to more than one alignment). That said I do tend to us ...


7

There are also third party free and open source basecallers that haven't been developed by Oxford Nanopore. Of particular note is Chiron, which gave the best uncorrected assembly identity among the base callers that Ryan Wick tested. It's slower than Albacore, but appears to be more customisable, and could theoretically be used to model any DNA feature that ...


6

Yes, you can click "stop acquisition" and your run won't be negatively affected. All of the reads are saved as they are generated. I am not sure how this will impact live basecalling though if that is something that you do.


5

In short, yes offline basecalling is supported. Oxford nanopore has made available an official offline basecaller, called albacore. If you have an account to the nanpore community website you can download it from here. Concerning running it in the field, last time I checked live basecalling was still requiring a lot of computing power, so you might want to ...


5

First of all - yes, you can generate FAST5 files and basecall later. Basecalling during the sequencing run is useful if you want results more quickly. You can also recall your FAST5 files with multiple basecallers, if desired. There are several ways to basecall currently: MinKNOW Albacore Guppy MinKNOW uses an embedded version of Albacore to perform its ...


5

It's worth noting two things as of Dec 2018: Albacore is being deprecated (but is still available from the Nanopore developer portal). Guppy is under active development, so Ryan Wick's comparisons may not reflect the current state of things. (The claim is that the current, just-released version of Guppy uses a new "flip-flop" algorithm that improves ...


4

No, poretools does not do basecalling. The poretools fastq command can be used to extract the FASTQ information from the basecalled FAST5 file (via MinKNOW live-basecalling or albacore). Alternatively, both of these basecallers can export a FASTQ file directly if desired. The difference between a basecalled FAST5 and a nonbasecalled FAST5 is the presence of ...


4

Yes, that should be fine, especially if basecalling is disabled. Locking a screen should be fine as well. I prefer to do basecalling after a run anyway, because there's a chance that it could chew up too much processor time and result in lots of skipped reads.


3

This has been set up as a bioinformatics protocol on protocols.io. I've been using LAST to identify the adapter orientation relative to the genome, and then using that information to split BAM files up and recombine them to create two strand-specific files that are displayable in a genome browser. I have written my own script to process LAST results into a ...


3

That's indeed the number of reads and that's quite low. How was your pore occupancy (number of pores sequencing) and your flow cell QC (number of pores good enough for sequencing)? How was your library concentration? Events is an approximation of nucleotides: it's a guess based on the current signal. As far as I know if you multiply this by 1.8 you get ...


3

There are actually 2048 usable sequencing wells, hexagonally packed with four wells connected to the same sequencing sensor/channel via a multiplex (mux) selector. The combination of the mux and the channel number determines the physical location of the well. Unfortunately, the association between channel number and physical location is not obvious, and ...


3

130k * 20bp is a small data set. At this scale, SSE2 Smith-Waterman may work well: git clone https://github.com/attractivechaos/klib cd klib && gcc -O2 -D_KSW_MAIN ksw.c -o ksw -lz ./ksw -a1 -b1 -q1 -r1 -t14 20bp.fa 20bp.fa > out.tsv I simulated 130k 20bp reads from E. coli K-12. The last command line takes about ~2 CPU hours based on the ...


3

My semi-automatic workflow for demultiplexing nanopore reads using LAST can be found here - pay attention to the comments, where I've identified bits in the steps which might trip up people trying to follow them: https://dx.doi.org/10.17504/protocols.io.7vmhn46 While this protocol is designed around using the ONT barcodes, the barcode fasta file can be ...


3

There are two other ways in which you could demultiplex reads if guppy doesn't have the config file for the kit you are using. Minibar: This works for dual index barcodes specifically Porechop: This is intended for the ONT sets, but you can also replace them with your own. However, it is designed to work with single index barcodes. That means, you will have ...


3

The classification is indeed based purely on comparing a mean to a threshold... but it's not the mean of the Phred scores that represent base quality on a logarithmic scale, but rather the mean of the underlying quality values prior to being transformed into Phreds. If I compute the "mean" quality for a read in this way - by converting each Phred in the ...


3

Use kraken2 with one of the 16S databases. Kraken2 has worked well for me with nanopore sequences. Pavian can be used to inspect the Kraken2 report files visually. Using uncorrected called reads has worked for me. Any errors from nanopore sequencing should be inconsistent enough (i.e. not always lead towards the same wrong organism) that the noise doesn't ...


2

For this application, you could probably also do something like calculate the Hamming distances between all of the strings in an all-vs-all approach (it should not take too long or too much overhead). You could use something like the Hamming distance tools in Julia. Here is an example of what I mean (using Julia): using StringDistances k = ["AATTGGCC", "...


2

Some replies from ONT is: OS X is supported and you can view the installation instructions on our Downloads page linked below. At the moment High Sierra is not officially supported as we have not yet fully tested it, but other users in the community have reported no issues with it so far. The compatibility test only performs a simplistic check ...


2

Not sure about conda as I don't use it, but I can highly recommend pipenv which is the python.org recommended package management system for python. I have tested the following on my Mac and it worked like a dream mkdir albacore && cd albacore mv ../ont_albacore-2.1.10-cp36-cp36m-macosx_10_11_x86_64.whl . # install pipenv if you don't have it ...


2

I found a page explaining where this is set up in a user_conf file here: https://community.nanoporetech.com/support/faq/test1/minknow/minknow/how-do-i-change-the-directory-for-the-reads-folder?search_term=user_conf Windows Open Notepad with administrative rights (search for notepad, right click and then select run as administrator). Select open file and ...


2

Probably not. ENA doesn't have any ability to specifically flag a file as 2D, and the nanopore file formats have changed a lot, so you pretty much need to rely on the submitter to separate out the reads in a meaningful way. Also, there wasn't really any consistent convention for what ONT used in their read headers. Looking at some of my old files they had a ...


2

I would keep it, in case you need to do another configuration later on. I have used mine for that purpose. I think storage doesn't really matter, I just put it in a drawer somewhere. Keep it away from liquids and extreme temperatures :)


2

My bet is that the nanopore folks have (of course!) done a ton of optimisation on two key things: The DNA cleanup The library preps I think that for most DNA extractions cleanups can vary a lot, but something like a Blue Pippin will do an exceptional job if you have access to one and can use it (they won't work for SUPER long DNA fragments which can't move ...


2

This one-liner below will work better for long reads than samtools flagstat in that it only counts the primary alignment for each read and samtools flagstat doesn't seem to calculate some stats for long reads. I have never seen samtools flagstat output stats on a per-reference basis, but am curious if so! This answer filters out secondary and supplementary ...


2

The MinION should be able to sequence DNA fragments entirely, regardless of the size, if you can get them to the pore intact. That is the problem: during DNA extraction and library preparation you will introduce breakage.


2

Yes, there is a tool to do this called R2C2 by the Vollmers lab at UC Santa Cruz. https://www.biorxiv.org/content/early/2018/06/04/338020


2

I would second the answer gringer gave recommending Kraken2. I would suggest using bracken to process the results from Kraken2 if you want to estimate species abundance (the raw Kraken2 results should not be used for this). I would recommend Pavian for visualising the results, it produces beautiful Sankey plots in a fairly pain-free manner. If you want to ...


2

The Guppy basecaller itself now has the ability to internally perform multiplexing. That is currently the preferred option. If for some reason, you still want a stand-alone application to demultiplex already basecalled reads, then qcat is an option: https://github.com/nanoporetech/qcat


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