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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21 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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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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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
35 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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110 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
200 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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22 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
16 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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1answer
23 views

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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66 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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3answers
118 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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72 views

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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99 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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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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67 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
161 views

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
212 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
60 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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265 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
162 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
129 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
169 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
98 views

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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2k views

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
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
585 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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72 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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33 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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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
370 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
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
367 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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329 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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671 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
433 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
171 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
230 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
70 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
655 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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698 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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548 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
2k 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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104 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
1k 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
932 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
168 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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351 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 ...