Questions tagged [pacbio]

Pacific Biosciences is a biotech company selling a third-generation sequencing technology often denoted by the same name, also known as Single-Molecule Real-Time (SMRT) sequencing. Questions related to data generated by PacBio/SMRT sequencing.

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Which quality score encoding does PacBio use?

Do you know which quality score encoding PacBio uses now? I know some of their file formats have changed in the past year or two, but I haven't found much on their quality score encoding. The most ...
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9 votes
2 answers
268 views

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 votes
2 answers
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estimate genome size: kmer-based approach from PacBio reads

Can anyone suggest a software/method for kmer analysis using PacBio reads (RSII)? Something similar to Jellyfish, that I saw in a nice tutorial - but must be suitable for long, noisy reads. ...
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7 votes
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: $$C = L*N / G$$...
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6 votes
2 answers
147 views

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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6 votes
1 answer
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What is the current state-of-the-art in assembling hybrid transcriptomes?

We are considering attempting de novo assembly of a species transcriptomes (i.e. without a reference genome) using the combined NGS outputs of Iso-seq and Illumina. One example I saw (Li et al 2017),...
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5 votes
1 answer
149 views

Why is a PacBio read length larger than the aligned reference region?

I recently had some Iso-Seq sequencing done on my organism catfish on the new Sequel platform and got weird alignments for a size selected 4 Kilobase and up fraction after running the isoseq3 pipeline....
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5 votes
1 answer
100 views

Can pair end reads with high heterozygosity be used to polish PacBio assembly?

I have used canu (correct) and smartdenovo to assembly PacBio reads. Next I am going to polish my assembly using illumina pair end reads by Pilon. However, I found there is high heterozygosity in my ...
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4 votes
2 answers
352 views

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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  • 601
2 votes
3 answers
195 views

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 ...
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2 votes
1 answer
620 views

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 ...
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2 votes
1 answer
229 views

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 ...
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2 votes
1 answer
52 views

polishing assembled genome to QV50 value

I am trying to assemble some genomes from pacbio reads using HGAP3 followed by quiver polishing. However, even after multiple rounds of quiver polishing, I cannot attain quality value 50 (QV50) for a ...
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2 votes
1 answer
149 views

Is it possible to find out the PacBio chemistry from the PacBio Sequel BAM's header?

Is it possible to find out the PacBio chemistry from the PacBio Sequel BAM's header? E.g. whether it was produced with P5-C3 or P6-C4? I checked mine but I haven't found it but maybe it's hidden in ...
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2 votes
0 answers
26 views

Under what circumstances can the SEQ field in a SAM file be a *?

I saw in the SAM format specifications that the SEQ field (10th column) can be a "*" if the sequence is not stored, instead of being the sequence of the mapped read. Under what circumstances ...
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2 votes
0 answers
214 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
34 views

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

I have two sets of PacBio reads from genomic DNA of an Aspergillus species that were made from separate preps of the culture. One of them has two additional peaks at 38% and 60% in the percent GC ...
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1 vote
1 answer
85 views

Best way to "fish" long reads that match a query sequence

Very simple set-up: We have a PacBio long high-fidelity (HiFi) reads, genome sequencing, and some of those that contain a particular sequence. I want to find out which ones. The length of the query ...
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1 vote
2 answers
215 views

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 ...
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1 vote
1 answer
77 views

Gap-fill assembly with PacBio reads

I would like to gap-fill (or correct) a mammalian-sized assembly for which we have BACs that have been sequenced with PacBio CLR data. I have seen there are pbm22/gcpp tools available for correcting ...
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1 vote
1 answer
61 views

How many reads do I need for hybrid assembly

I have Illumina and PacBio reads and I would like to use dbg2olc for hybrid assembly. Part of dbg2olc is SelectLongestReads which select reads that sum to ...
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1 vote
0 answers
17 views

Why does gene count increase drastically after scaffolding with Hi-C data?

I have a conceptual question. I have a diploid, outcrossing plant genome assembly of ~1.1 Gb size. The original assembly is generated from PacBio reads. After genome annotation with the ...
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0 votes
3 answers
265 views

How to install blasr on Mac OS X?

I am trying to install blasr on mac osx. I followed the steps here I get the error below after using the 'make' command: ...
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0 votes
2 answers
34 views

How do you set the coverage in PacBio's Sequel II?

I am reading the Whole Genome Sequencing for de novo Assembly Best Practices Use the Sequel II or IIe System and SMRT® Cell 8M to sequence to desired coverage depth for complexity of genome 10- to 15-...
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0 votes
1 answer
37 views

How to improve a genome assembly using Dovetail and PacBio assembly?

I have more of a conceptual question. I have two genome assemblies from the same plant, one from Dovetail technology (~998 Gb) and another is PacBio HiFi assembly (~1.1 Gb). The Dovetail assembly is ...
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0 votes
1 answer
99 views

How to de novo hybrid assemble with Pacbio CCS and Illumina PE reads

I would like to perform de novo genome assembly on a diploid microalgal strain. I have two datasets: PacBio CCS/HiFi reads, low coverage. Illumina PE 2x150 (standard shotgun) Does anybody have any ...
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0 votes
1 answer
64 views

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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0 votes
0 answers
29 views

How to manually curate a genome assembly for sequence variation or error?

I have a PacBio HiFi assembly of 1.1 Gb from a heterozygous species. I have aligned this assembly against a reference genome which is around 0.9 Gb. I can see that there are quite a few INDELs, ...
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0 votes
3 answers
65 views

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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0 votes
0 answers
69 views

Required input files for StringTie2

I am working on a virtual project for WGS combined with RNA seq for annotation. The RNA will be sequenced using PacBio Isoseq (Sequel II, HiFi-reads). With some research, I found that StringTie2 is ...
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