Questions tagged [long-reads]
Questions relating to long reads, such as pacbio or nanopore reads.
45
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How to deal with heterozygosity during polishing of genome assembly based on long reads?
All the long-read sequencing platforms are based on single-molecule sequencing which causes higher per-base error rates. For this reason a polishing step was added to genome assembly pipelines - ...
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1
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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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16
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2
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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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9
votes
2
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calling diploid SNVs from long reads
I'd like to call diploid SNV variants from long-reads data (~80SMRTcells PacBio).
I have generated a draft reference genome for an indivudual from a heterozygous (~4%) species (Canu+Haplomerger2).
...
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8
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4
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What reasons are there to choose Illumina if PacBio provides longer and better reads?
PacBio provides longer read length than Illumina's short-length reads. Longer reads offer better opportunity for genome assembly, structural variant calling. It is not worse than short reads for ...
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8
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1
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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 ...
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8
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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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7
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1
answer
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Coverage calculation: long reads (RNA-seq)
Say your aim is to calculate the coverage of an RNA-seq experiment generated with long-read sequencing (so, uneven read length).
Up to now, I relied on the Lander/Waterman equation:
$$C = L*N / G$$...
- 2,656
7
votes
1
answer
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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 ...
- 5,060
6
votes
1
answer
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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 ...
- 1,059
6
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2
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Structural variant calling for low-coverage PacBio data
PacBio is selling ~10x PacBio SEQUEL long reads as an upgrade to Illumina data for SV discovery.
In a clinical setting, the main requirements are proper sensitivity and specificity but also the ...
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2
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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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5
votes
1
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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 ...
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4
votes
2
answers
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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 ...
- 2,459
4
votes
2
answers
272
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Sort reads in BAM file based on presence of specific deletion?
I have an indexed BAM file containing long-read sequencing data and I'd like to split the reads contained within into those with a known deletion and those without the deletion (I have the deletion ...
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4
votes
2
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Mapping Reads to Known Gene Paralogs with Long Read Technology
I have some sequencing data from a captured region that is a known paralog edited. For now, I have been mapping the data using standard minimap2 flags for PacBio DNA sequencing:
...
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4
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1
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How to map short sequences to long reads, recovering all multiply-mapped high-quality matches
The dilemma:
I have around one million short sequences (21 bp to several 100s of basepairs) for which I need to identify all occurrences of in 20-30x coverage noisy long reads (both pacbio and ONT). ...
- 3,141
4
votes
1
answer
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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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4
votes
1
answer
67
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Tree Building Algorithm that treats gaps as deletions
I'm part of a nanopore sequencing experiment that will sequence several generations of viruses. The intent is to perform directed evolution by putting selective pressure on these viruses and tracking ...
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4
votes
1
answer
317
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Find pattern that is present twice and allow <=2 mismatches on each
I have a fastq file of 400,000 reads (so speed is important). In the sequences there are barcodes integrated that should be present twice. Given a barcode, I want to find the sequences that have the ...
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3
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1
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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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3
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1
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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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3
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1
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Scaffolding a genome with hybrid data
I am assembling a ~500MB genome, and have ~150x long reads and ~200x 150bp PE short reads, with a ~400bp insert size.
I've done a lot of work with minimap+miniasm, and have what I think is a good set ...
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3
votes
0
answers
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Can multiplexing in Sequel II SMRTcells reduce the coverage?
I would like to have high depth of coverage of the transcriptome, but I need to analyze several tissues. I was suggested to pool all 5 tissue samples in same SMRT 8M cell. Will this result in a ...
- 307
3
votes
1
answer
259
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Chromosomal order of supplementary alignment in BAM file "SA" tag
I have several long-read sequences, which when aligned to a human reference genome, aligns to multiple chromosomal fragment as a result of chromothripsis shattering the linear DNA and randomly piecing ...
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2
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3
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238
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What assembler is appropriate for High-Fidelity PacBio reads
What assembler is appropriate for High-Fidelity PacBio reads?
For example, canu is good for high-error PacBio reads. But what algorithm to use for HiFi reads? Would it be OK to use canu without the ...
- 2,459
2
votes
1
answer
138
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PacBio long-reads impact in transcriptome de novo assembly?
We are strongly interested in assembly a good transcriptome of reference for a non-model organism and build a local database. We have sequenced the same individual with Illumina (150 millions of pair-...
2
votes
1
answer
393
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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 ...
- 283
2
votes
2
answers
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Calling isoforms from long read data generated from partially degraded RNA
What will be the best tool to call isoforms from long read data generated from partially degraded RNA. By mistake we processed some samples with poor quality RNA to generate long read. Now we are ...
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2
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1
answer
425
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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, ...
- 3,141
2
votes
1
answer
686
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Can I pilon-polish long reads with Illumina short reads to improve structural variant detection?
I have pacbio Sequel data at 50 x coverage for a strain of animal. I would like to find structural variants compared to the reference genome sequence. At the moment, I align my reads to the reference ...
- 2,459
2
votes
1
answer
265
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How to interpret the SMRT Link base modification algorithm output?
I have got PacBio data from a Sequel machine at a coverage > 50X. I have run it in SMRT Link with their Base Modification Detection algorithm. I am now trying to figure out how to interpret the output ...
- 2,459
2
votes
1
answer
1k
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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 ...
- 2,459
2
votes
1
answer
108
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somatic SNV tool for ONT samples
I'm looking for a tool to call somatic single nucleotide variations on reads from an Oxford Nanopore Technologies (ONT) run.
I have both tumor and normal samples and have already aligned them to a ...
2
votes
1
answer
499
views
Mark BWA-SW split alignments in output for long reads
Is there a way to remove split alignments of single-end long reads from BWA-SW output? BWA-MEM has an option to include or flag split alignments in the result, but it seems BWA-SW method include them ...
2
votes
0
answers
332
views
Platanus-allee phasing fail: Error(13): Error, SolveDBG exception!
I am using Platanus-allee 2.2.2 for heterozygous genome (~500mb) assembly with Illumina short reads and PacBio reads input data.
I have the file contigs.fa from short reads but phasing step with ...
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2
votes
1
answer
83
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Why does samtools mpileup sometimes include ref bases (other than ',' or '.')?
This is my first post here. I can think of no way of giving an easily reproducible example, as per the stack ethos. SO apologies in advance and any feedback on question format appreciated.
I have ...
- 579
1
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1
answer
837
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Difference between paired-end, mate-pair and long read
I writing here because I have some questions for you.
I wondered what the essential differences were between paired-end, ...
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1
vote
1
answer
79
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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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vote
1
answer
250
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What is a PacBio "movie file"?
I came across references to "movie files" from PacBio sequencing in this paper:
https://www.jimmunol.org/content/204/12/3434
Specifically:
Movie files used to generate results presented in ...
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1
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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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1
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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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2
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Is it possible to filter contaminated reads for raw PacBio sequences (not HiFi reads) before assembly?
De novo genome assembly for non-model organisms face the issue of bacterial contamination. For assembled contigs with mostly bacterial-like sequences (based on BLAST search), the entire contig can be ...
1
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3
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381
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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 ...
- 2,739
0
votes
1
answer
659
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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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