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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Polishing PacBio or ONT with Illumina

Which tool would be good to polish PacBio or ONT with Illumina? Thank you in advance
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NanoPlot Error : Kaleido Problem

I'm trying to visualize my fastq file using NanoPlot 1.38.1, but it ends up with this error in the log file: ...
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1answer
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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
24 views

How do I tell that a MinION run has finished based on the files produced?

I want to automate some scripts which should run after a MinION run completes. Is there a file which is produced by MinKNOW which will enable me to tell that the run has finished?
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Split FASTQ and matching BAM into matching chunks

I am running a slow downstream analysis on a large set of nanopore reads (approx 3 million), and would like to split them into smaller chunks, run the analysis in massively parallel, and then ...
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Doing functional enrichment of microbiome data

I have done gene set enrichment analysis which is now straight forward. Such as taking gene set and making a query. For microbiome data it is not that straight forward which I found out. Step done so ...
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7answers
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Read length distribution from FASTA file

I have a single ~10GB FASTA file generated from an Oxford Nanopore Technologies' MinION run, with >1M reads of mean length ~8Kb. How can I quickly and efficiently calculate the distribution of read ...
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1answer
136 views

What are some ways to error correct Oxford Nanopore long read sequencing?

I am sequencing long read genomic sequences to assemble MHC region haplotypes in a non model organism using Cas9 Sequencing Kit (SQK-CS9109) using flongle adaptors in the minION from Oxford Nanopore ...
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1answer
37 views

Detecting SARS-CoV-2 variants from the mixed virus population

I have a fastq file from Nanopore sequencing data that contains reads from the UK and South Africa variants of SARS-CoV-2. These variants are identified by three key mutations in the receptor-binding ...
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1answer
79 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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1answer
35 views

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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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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3answers
136 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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1answer
202 views

Oxford nanopore promethion; does guppy3 separate fast5 into pass and fail? If not what does?

just a quick question about fast5 pass fail folders. I have been working under the assumption that the fast5 pass fail folders I was given as raw data from our ONT vendor came from them doing base ...
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1answer
255 views

Upgrading guppy basecaller on Oxford Nanopore Gridion via PPA

I am trying to update the guppy_basecaller (current version installed is 3.2.10+aabd4ec) on a ONT Gridion device. I am initially trying to update it via the ...
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1answer
25 views

Nanopore data clarification regarding merging of samples

We had same set of sample ran on nanopore platform twice. The issue on hand is. First run was done it was nearly done but the power went off. Second run was done completely with enough sequencing ...
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1answer
337 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
17 views

ambiguous calls in nanopolish

I am converting scores into frequencies (from nanopolish results) using Nanopore scripts. The script filters out lots of rows on the premise that reads where ...
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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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341 views

Finding the location and unit length of repetitive sequences within a long read

After discovering a few difficulties with genome assembly, I've taken an interest in finding and categorising repetitive DNA sequences, such as this one from Nippostrongylus brasiliensis [each base is ...
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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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2answers
283 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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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
135 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
609 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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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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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
233 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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2answers
2k 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
62 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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4answers
682 views

Tools for simulating Oxford Nanopore reads

Are there any free open source software tools available for simulating Oxford Nanopore reads?
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0answers
137 views

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
185 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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1answer
203 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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725 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
1k 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
76 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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1answer
438 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
2k 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
377 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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1answer
1k views

STAR-long parameters for aligning RNA ONT reads to genome

Are there any suggested parameters to align ONT reads to the reference genome using STAR-long? For now, I used the parameters suggested here, but I noticed a weird behaviour. I have RNA reads (D. ...
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1answer
425 views

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
696 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
180 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
564 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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4answers
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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
547 views

How can I improve a long-read assembly with a repetitive genome?

I'm currently trying to assembly a genome from a rodent parasite, Nippostrongylus brasiliensis. This genome does have an existing reference genome, but it is highly fragmented. Here are some ...
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1answer
943 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
231 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 ...