Questions tagged [nanopore]

Questions specific to nanopore sequencing. For general question about long reads, use tag long-reads instead and for questions about specific sequencer use a specific sequencer tag (i.e. minion, gridion)

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What is the typical host-to-bug DNA ratio found in nanopore sequencing without amplification?

I'm interested in sequencing a sputum sample using an ONT MinION without performing any type of DNA amplification. Has anyone found a good reference (or anecdotal evidence) for a range of human to ...
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1answer
36 views

Master of Pores Installation Mac OS Mojave 10.14

I'm bit of a newbie with ONT analyses and am having some trouble with the Master Of Pores installation instructions (https://biocorecrg.github.io/master_of_pores/install.html). I've managed to install ...
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2answers
51 views

Recommended workflow for RNA long read Kallisto-like TPM estimation?

On short reads, Kallisto/Salmon is a standard workflow for measuring RNA transcript counts in TPM. However, when I tried to Google a similar workflow for long reads, it's not clear there is a ...
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How do I find all supplementary mappings of a chimeric long (ONT) read mapped with NGMLR?

Unlike this guy How do I find split reads? I have done a lot of reading before asking this question. I'm aware that the FLAG will tell me if a read is supplementary and the CIGAR has information ...
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nanopore QC measures on fastq file

I am working with virus nanopore sequencing data. I have to remove host genome from the reads. I want to run the QC on the fastq file after host genome subtraction. I have used nanoplot. It is slow. I ...
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Nanopore microbiome data analysis 16s rRNA data

The steps i have done so far used guppy to do base calling from fast5 to fastq so what i see is in each barcode file i have 1960 fastq files which i have merged. The i did trim the fastq files using ...
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1answer
45 views

snakemake - using output file not explicitly declared as input for subsequent rule

I am writing a snakemake that, up to now should use raw sequencing reads to produce a draft assembly using canu use minimap2 to align the raw reads against the draft assembly, generated by canu. ...
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1answer
51 views

Assembly reads having a copy of their beginning in their tail

I am analyzing the reads for the SARS-CoV-2 assembly with id SRR11140748. Apparently these reads were obtained with parallel sequencing by Illumina and Oxford Nanopore Technologies. I have found these ...
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Train a new basecalling model for nanopore?

Basecalling on nanopore requires a machine learning neural network model, for example here A popular software from Nanopore for it is https://github.com/nanoporetech/taiyaki, which has many people ...
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2answers
47 views

Program Quality control for data sequencing (nanopore)

Hope all of you are fine. I am wondering if exists a program easy to use that can bring me Basic Statistics of Quality Control from raw data made by MinION device (nanopore). I have read about ...
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1answer
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Kallisto for Nanopore?

Although the Kallisto software was designed for RNA, it has also been very reliable in counting selected FASTA sequences in DNA sequencing. For example, given a WGS sample and my FASTA file. Kallisto ...
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1answer
108 views

nanopore - where to retrieve information from the basecaller used

Where can I find information about the basecaller used in a specific nanopore run (we have a Gridion machine)? I know already that we have Guppy v. 3.0.3, which I ...
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1answer
52 views

How do you map nanopore fast5 files?

Is there a best practices for mapping Oxford Nanopore files to a reference? Is there a tool that can take a tarball of fast5 files and map them directly or do they need to be converted to fastq first?...
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189 views

Microbial Classification with 16S Nanopore Data

I have recently been tasked with analyzing a heterogeneous microbial sample composed of 16S data generated on a MinION sequencer, with the goal of assigning taxonomies and calculating respective ...
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1answer
102 views

BED file from .bam alignment structure

I am using a pipeline from nanopore to detect structural variants (SVs) in a human sample with long-reads sequencing. The first steps of the pipeline are: index the reference genome with ...
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0answers
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Nanopore FASTQ header specifications

Is there a specification anywhere for how a Nanopore FASTQ header should be formatted? I am creating strategies for generating test FASTQ files and would like to ensure I am generating "correct" ...
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1answer
111 views

Oxford Nanopore mapping Quality and sequencing error

I'm analyzing some Oxford Nanopore sequencing reads, with several articles stating the sequencing error is between 5% and 15%. Since its 1D and not 1D^2 sequencing i would assume the sequencing error ...
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3answers
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Compare multiple alignment results' aligned bases

I have aligned a nanopore data set to a reference genome with graphmap, minimap2 and BLASR. The alignment results are stored in BAM files. I would like to do some concordance assessment, looking at ...
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2answers
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Error while calling bcftools mpileup - Failed to open -: unknown file type

I have sequenced a bacterial genome with a GridIon from ONT. Basically what I want to check is whether or not trimming 50 bps at the beginning of the reads will improve alignment against the reference ...
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1answer
602 views

How does MinKNOW classify 1D reads as “pass” or “fail”?

I have found a couple of sources1,2 that indicate that a read in a 1D² run is classified by MinKNOW as "pass" and put into the fastq_pass folder if both of the ...
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1answer
423 views

How do you normalise read coverage in a BAM file?

This is a question from the Oxford Nanopore community, from user Michael Radzieta: I have sequenced some plasmids using the rapid barcoding kit, I have attempted to assemble the data using several ...
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1answer
59 views

What is a signal 'chunk' in the context of Nanopore sequencing?

Does one chunk correspond to the raw signal of a single read read? Why does messing with this parameter affect basecalling results?
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Help with 1D^2 library shearing

I've done my 1st trial of 1D^2 whole genome sequencing using LSK-SQK308 kit with R9.5 flowcell. Without doing library fragmentation, I encountered the unexpected library shearing during sequencing. ...
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1answer
925 views

Significance and timing of “mux scans”

I'm using MinIONQC to do quality control on some ONT data. The software plots several read characteristics over time (hours passed during the sequencing process). These plots contain several vertical ...
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1answer
336 views

Is nanopolish worth it since faster polishing software is available?

For Oxford Nanopore contigs produced by any long-read assembler has anyone performed any benchmarks to compare the polishing tools racon, ...
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2answers
1k views

Demultiplex nanopore reads with custom barcodes

We have a problem trying to demultiplex MinION sequences with custom barcodes. Do you have any software recommendations we can try for demultiplexing or how to demultiplex these custom barcodes with ...
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1answer
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Questions regarding Nanopore sequencing analysis

I am new to Nanopore sequencing analysis. I have a couple of questions regarding it which are as follows: How do I know if my fast5 file is multiread or single read file? Is there a way to combine ...
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1answer
291 views

wtdbg2: practical implications of k-mer fsize and psize choice

I am using wtdbg2 2.3 to assemble a human genome (sequenced on PromethION from a cell line). I filtered out reads with low average quality, and now I am trying to determine the parameters that will ...
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1answer
561 views

Why do I get so many insertions from Minimap2 on my Nanopore WGS?

I'm a starting my analysis on nanopore whole genome sequencing. I start my analysis from this popular Github. The sample I downloaded was WGS for NA12878, so I would assume it's alignment to GRCh38 ...
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1answer
390 views

Albacore basecalling running but outputs 0 reads

I am trying to basecall data produced by the MinION using the SQK-LSK109 kit and FLO-MIN106 flowcell via the command-line. My version of albacore is the latest (v2.3.4). I tried running using the ...
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1answer
155 views

Question on nanopore sequencing data process pipeline (cDNA-PCR)

I recently started doing the analysis on nanopore sequencing data. As I was searching for some help on pre-processing of the data, I found your nice setup pipeline created here: https://www.protocols....
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1answer
222 views

Error correction within the long read

I am going to get some data from plasmid sequencing to identify SNPs on the plasmids. What is done in the lab is the following: The plasmids are purified by size. We amplify the plasmids using the ...
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1answer
69 views

Using ONT MinION, why is that we cannot get a full length DNA read?

Here, https://www.youtube.com/watch?v=E9-Rm5AoZGw, ONT claims they can read non-fragmented DNA and that with the hairpin like structure the double strands can be read at once. Then why is that MinION ...
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3answers
575 views

Total reads aligning to each reference within a bam file

I have two PCR amplicons that have been multiplexed and sequenced using the nanopore minion. I have aligned the fastq reads using minimap2 with a reference file containing both amplicon sequences and ...
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2answers
587 views

How to simulate nanopore reads?

I have looked already here: Tools for simulating Oxford Nanopore reads . This doesn't answer my question, because it lists a few Nanopore read simulators, but I have specific problems with each of ...
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1answer
501 views

Extracting strand specific reads from MinION cDNA-PCR protocol

I recently performed my first MinION run, and now I'm trying to analyze the data. Being pretty new to the bioinformatics field, I was hoping some of you could help me out. As a bit of background, I'...
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2answers
1k views

Can I stop my nanopore sequencing run if there are no more reads being produced?

I am sequencing a whole genome on MinION. I have used this flow cell for 6 hours for the phage lambda control. That gave me around 300,000 reads. I started a 48h sequencing run on the same flow cell ...
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0answers
99 views

aligner for 1D^2 oxford nanopore data

I am wondering if anyone has tried aligning 1d^2 RNA-seq data. I am trying that on minimap2. It is giving me very low ~50% of mapped reads. it gives ~95% mapped reads for 1D. I am using the following ...
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1answer
872 views

How many reads has my sequencing run produced on minion?

I am running a 48 h sequencing protocol on a FLO-MIN107, on MinION, with DNA library-prepped with SQK-RAD004. MinKNOW is installed on my Mac and controlling the MinION. I have found the following ...
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1answer
861 views

Albacore wheel not supported on this platform (Mac OS X El Capitan)

I download the Albacore wheel from Oxford Nanopore. I activated my conda environment that has Python 3 as the default python version. I tried to install Albacore with ...
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1answer
165 views

Basecalling process carried out by full_1dsq_basecaller.py/.exe

I am unable to understand what exactly the full_1dsq_basecaller.py/.exe script does. Is it giving the consensus sequence for linear and complementary strand, or it is just giving the reads that came ...
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1answer
3k views

Set directory where MinKNOW writes FAST5 files

I would like to set the folder in which MinKNOW writes the raw data. How can I do this? I do not currently know where MinKNOW will output my data. What is the default directory where MinKNOW outputs ...
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2answers
283 views

Can I use my computer while MinION is sequencing without negatively affecting the run?

I have a Mac Book Pro and I am about to start a sequencing run on the MinION. MinKNOW is going to be running for 2 days. Can I use my computer during sequencing? Can I browse the web and use Excel, ...
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1answer
2k views

Difference between 1D and 1D^2 data

I am working on ONT data. Initially, I have worked on data from 1D using Minimap2 aligner. I came across 1D^2 from ONT website. I was wondering how different is the data from two techniques? What is ...
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1answer
377 views

What to do with the configuration test cell after configuring the MinION with it?

What to do with the configuration test cell after configuring the MinION with it? I received a MinION with a configuration test cell in it. After running a configuration in the MinKNOW software while ...
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4answers
5k views

What are the pros and cons of the different basecallers in Oxford Nanopore Technology Sequencing?

What are the pros and cons of the different basecallers in Oxford Nanopore Technology Sequencing? I am about to start a MinION run on my laptop. What should I consider when choosing my basecaller? ...
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1answer
573 views

Does MinKNOW work with Mac OSX high sierra 10.13.1?

I have done a compatibility check as described here on my computer to know if I can use MinKNOW on my mac laptop. It says that my OS is incompatible and that the OSX must be Yosemite or El Capitan. ...
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1answer
320 views

Removing repeated reads from nanopore 1D² reads

This is summary of discussions and a question that was posted by Devon O'Rourke on Twitter Following Albacore basecalls on a 1D² library I get two sets of .fq files, summary stats, etc.: one for the ...
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3answers
374 views

sort a fasta file containing the Oxford Nanopore Technologies (ONT) header by sequencing start_time ascending

I have basecalled ONT reads and converted them to multifasta. The multifasta contains the original ONT headers in this format: ...
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2answers
248 views

Canu assembly not making a single consensus?

I've downloaded reads from this BioProject. Using canu with default parameters (no correction), I've got 4 contigs, none of which really look like the reference plasmid here. The command I used was: ...