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16 votes
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What reasons are there to choose Illumina if PacBio provides longer and better reads?

There are so many reasons why one might want to prefer Illumina over PacBio (also note that it's a false dichotomy, at least Oxford Nanopore is a competitive sequencing platform): The first (IMHO and ...
12 votes
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Which quality score encoding does PacBio use?

PacBio does use PHRED 33, but it turns out the question may be irrelevant for the newer PacBio Sequel Sequencer, because it reports all base qualities as PHRED 0 (ASCII ...
Mark Ebbert's user avatar
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6 votes
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Coverage calculation: long reads (RNA-seq)

The read length is irrelevant when calculating the mean coverage statistic. It's simply the total number of bases sequenced divided by the target Xome length. In the example provided in the question, ...
gringer's user avatar
  • 14.7k
6 votes

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

I also wrote a package to create various plots from Oxford Nanopore sequencing data and alignments: NanoPlot. It can be installed through pip (see also the README on Github). In addition to multiple ...
Wouter De Coster's user avatar
5 votes

What reasons are there to choose Illumina if PacBio provides longer and better reads?

Many analyses performed on Illumina machines these days require large numbers of reads. For example, most analyses in ChIP-seq, RNA-seq, ATAC-seq etc, need 10s or even 100s of millions of reads for ...
Ian Sudbery's user avatar
  • 3,331
5 votes
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estimate genome size: kmer-based approach from PacBio reads

I don't think there is a method that would estimate a genome size using raw long reads. The genome size estimates based on raw reads are done by fitting a model to kmer spectra (for instance ...
Kamil S Jaron's user avatar
5 votes
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Why is a PacBio read length larger than the aligned reference region?

Q1 Probably not - this is probably just displaying the contents of the input (I presume a bam file) exactly as they are encoded. Q2 The splicing looks a little ...
conchoecia's user avatar
  • 3,181
4 votes

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

It's important to always consider read length and quality jointly with high-error read data, and current long-read technologies (e.g., MinION and PacBio) have high error rates. Considering read length ...
Mark Ebbert's user avatar
  • 1,374
4 votes
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What is the most accurate approach for de Novo sequencing?

HiFi reads can resolve most repeats in a genome. Illumina reads from these repetitive regions are likely to get mismapped and these mapping errors may become consensus errors. Most short-read ...
user172818's user avatar
  • 6,575
4 votes

how to see pacbio reads in IGV

You should just open the bam file, not one of the index files.
Wouter De Coster's user avatar
3 votes

What assembler is appropriate for High-Fidelity PacBio reads

As recently tweeted by A. Phillippy, Canu v1.9 now supports HiFi reads. Therefore you should just read their manual and look for the implementation :)
Felipe Almeida's user avatar
3 votes
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Best way to "fish" long reads that match a query sequence

I have seen the blasr aligner typically used for aligning PacBio reads to a reference. Is there a reason why blasr would not be a good choice? I guess the very short sequence you are trying to align ...
Jay Moore's user avatar
  • 1,012
3 votes

What reasons are there to choose Illumina if PacBio provides longer and better reads?

Three reasons for Illumina: Much better for a large number of samples (easily handle 96 samples). SNP calling is much better - much greater depth Hardware costs, an Illumina MiSeq machine is cheap ...
M__'s user avatar
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3 votes
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Mapping Reads to Known Gene Paralogs with Long Read Technology

Question 1 This command will get you all of supplementary alignments for the reads. This isn't exactly what you want though. You want all of the reads that have more than one mapping. ...
conchoecia's user avatar
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3 votes
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Can pair end reads with high heterozygosity be used to polish PacBio assembly?

Resequencing your sample is not going to make it any less heterozygous. I am assuming that you are working with a diploid organism? Please update your question and specify the genome size and ploidy ...
conchoecia's user avatar
  • 3,181
3 votes
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How to install blasr on Mac OS X?

The path of least resistance is probably to run the software inside a container. If you install docker, you can just do the following: ...
heathobrien's user avatar
  • 1,816
3 votes
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How to subset an SRA file for a single chromosome?

After dumping out the reads using fasterq-dump, you'll need to align them first and then extract those that map to your region of interest. I think minimap2 is an ...
Steve's user avatar
  • 3,118
2 votes

Structural variant calling for low-coverage PacBio data

There is an evaluation of PB Honey and Sniffles algorithms for low coverage PacBio datasets in this preprint and another evaluation is shown on this poster. Both reports agree that optimal is (...
Kamil S Jaron's user avatar
2 votes

How to install blasr on Mac OS X?

I've had many issues with installing blasr on my own, but it's a breeze using (bio)conda, see https://anaconda.org/bioconda/blasr conda install -c bioconda blasr
Wouter De Coster's user avatar
2 votes
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How to interpret the SMRT Link base modification algorithm output?

It sounds like you're looking for the modifications.csv file, rather than the modifications.gff file. The ...
Mark Ebbert's user avatar
  • 1,374
2 votes
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calling diploid SNVs from long reads

You may want to try the longshot tool (https://github.com/pjedge/longshot) developed for calling variants in diploid genomes from long read data.
user4162's user avatar
2 votes

calling diploid SNVs from long reads

You could try aligning your reads to the draft reference genome with for example minimap2 and calling variants with freebayes. ...
winni2k's user avatar
  • 2,286
2 votes

What assembler is appropriate for High-Fidelity PacBio reads

From the group of Heng Li there is hifiasm.
Wouter De Coster's user avatar
2 votes
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Can I pilon-polish long reads with Illumina short reads to improve structural variant detection?

I'd recommend using Canu for correction rather than Pilon, because it has a component that is specifically designed for read-level correction. The newest version of Canu (v1.7) will track all reads ...
gringer's user avatar
  • 14.7k
2 votes
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How do you set the coverage in PacBio's Sequel II?

They just want to know the ploidy of the sample. In a diploid sample, e.g. humans, there are two haplotypes. This is mostly important for haplotype resolution (i.e. get the two full genomes in a ...
Maximilian Press's user avatar
2 votes
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How can Iso-Seq reverse transcriptase artifacts be avoided?

It is possible to filter out iso-seq reads with RT artifacts, though I don't know how effective these informatics approaches are. With nanopore, you will get more false junctions in alignment or ...
user172818's user avatar
  • 6,575
2 votes

regex: samtools command to "refine" PacBio IsoSeq data?

The samtools view command you've provided just uses a filter expression to discard records that do not meet the specified expression criteria. In this case, the ...
Steve's user avatar
  • 3,118
2 votes
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Hybrid assembly versus polishing for hifi and illumina reads

Usually, if you have HiFi reads, there is no way you can improve the assembly with short reads. Not just that they don't have higher quality, but they also map a lot worse. People sometimes use short ...
Kamil S Jaron's user avatar
1 vote

polishing assembled genome to QV50 value

15 times is too many times. If you were going to get quiver to get you good QVs it would have happened after 2-3 rounds probably. Some questions: Why are you using quiver? Are your reads from RSII ...
Maximilian Press's user avatar
1 vote

Do additional peaks in percent GC of PacBio gDNA reads indicate contamination?

I would suggest plugging your reads into a fast k-mer matching tool like mash screen to get a basic idea of where your reads come from, if you suspect contamination. An issue with just looking at GC ...
Maximilian Press's user avatar

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