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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26
votes
7answers
7k views

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 ...
21
votes
4answers
665 views

Tools for simulating Oxford Nanopore reads

Are there any free open source software tools available for simulating Oxford Nanopore reads?
17
votes
1answer
541 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 ...
13
votes
1answer
205 views

Compare alignment quality of multiple sequencing runs aligned against the same reference genome

I have run Oxford Nanopore Technologies' MinION sequencing on the same DNA sample using three flowcells, each aligned against the same reference genome (E.coli K12 MG1655) using both BWA MEM and ...
13
votes
2answers
329 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 ...
12
votes
2answers
2k views

How to convert fastq to fast5

fast5 is a variant of HDF5 the native format in which raw data from Oxford Nanopore MinION are provided. You can easily extract ...
9
votes
3answers
542 views

Visualisation of long read RNA-Seq splicing

I have a dataset of Oxford Nanopore cDNA reads. Many of my reads are full-length or close to full-length transcripts, and I and am interested in examining alternative splicing. For this, I would like ...
9
votes
2answers
2k views

How do you generate read-length vs read-quality plot for long-read sequencing data (e.g., MinION)?

How do you generate read-length vs read-quality plot (heat map with histograms in the margin) for long-read sequencing data from the Oxford Nanopore Technologies (ONT) MinION? The MinKNOW software ...
8
votes
4answers
6k 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? ...
8
votes
2answers
538 views

Building STAR Genome Index for nanopore RNA sequencing

I am aligning a dataset of 1,000,000 reads oh human mRNA sequenced on Oxford Nanopore Technologies' MinION, and would like to use the STAR aligner, using the parameters recommended by Pacific ...
8
votes
4answers
99 views

Introduce errors in reference transcripts according to external dataset error model

I would like to modify some reference transcripts from Ensembl (D. melanogaster) to introduce a controlled rate of random errors in the sequences. The idea would be to introduce random base ...
8
votes
1answer
888 views

How can I use Nanopore reads to close gaps or resolve repeats in a short-read assembly?

Low coverage MinION reads should be useful to close gaps and resolve repeats left by short-read assemblers. However, I haven't had any success with the software I know about. I'm aware of the ...
7
votes
3answers
762 views

Extract nanopore read ID & start times from fastq file

I have a fastq file from minION (albacore) that contains information on the read ID and the start time of the read. I want to extract these two bits of information into a single csv file. I've been ...
7
votes
2answers
253 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: ...
7
votes
1answer
387 views

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 ...
7
votes
1answer
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'...
7
votes
1answer
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 ...
6
votes
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 ...
6
votes
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 ...
6
votes
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. ...
6
votes
1answer
364 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 ...
6
votes
1answer
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. ...
5
votes
2answers
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 ...
5
votes
2answers
350 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, ...
5
votes
2answers
196 views

How to estimate whether a long-read is meaningful sequence?

The setup Imagine that I work on an organism without a reference genome, and that the closest reference genome I can get is quite diverged. E.g. ~10% diverged in terms of SNVs when measured with ...
5
votes
1answer
200 views

Detecting structural variants with MinION data

Working on various cancers I have an interest in detecting structural variation (SV) in human, we've successfully used various tools like Pindel, SVDetect, Manta, and LUMPY, to name a few for ...
5
votes
1answer
205 views

Rapid metagenomics classifiers on long read data [closed]

I recently used the minION (Nanopore, 9.4 flow cell, RAD001 kit) to generate a metagenome out of environmental samples. Passed reads weren't brilliant (196, average 1,594bp lenght), but working with ...
4
votes
1answer
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 ...
4
votes
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 ...
4
votes
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 ...
4
votes
2answers
1k views

How to get Nanopore MinION fast5 from SRA

I found some Nanopore MinION data on SRA, which I would like to investigate. I use sratoolkit for Illumina data all the time, but I am not sure how to get the ...
4
votes
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 ...
4
votes
2answers
397 views

Error rate setting in Canu error correction

I want to use Canu to correct my nanopore long read (version: MinION R9.5), but I am not quite sure how to set the correctErrorRate. Should I follow the Canu manual (Nanopore R7 2D and Nanopore R9 1D ...
4
votes
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 ...
4
votes
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....
3
votes
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 ...
3
votes
2answers
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 ...
3
votes
3answers
428 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: ...
3
votes
2answers
444 views

Minion channel ID's from Albacore

The latest version of Albacore from Oxford Nanopore Technologies calls bases from raw fast5 files. A useful piece of output is the sequence_summary.txt, which is a ...
3
votes
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 ...
3
votes
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 ...
3
votes
2answers
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 ...
3
votes
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 ...
3
votes
1answer
651 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. ...
3
votes
1answer
344 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 ...
2
votes
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 ...
2
votes
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, ...
2
votes
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 ...
2
votes
1answer
481 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 ...
2
votes
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" ...