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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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Hybrid assembly versus polishing for hifi and illumina reads

I will have to carry out a project of assembly using hifi reads for which I have already illumina reads and I am wondering which of the hybrid assembly or polishing would be the best option for this ...
R-addict's user avatar
1 vote
1 answer
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ipdSummary command stops working if --identify m5C_TET is included in command, and does not give any m5C info either

I have installed the latest SMRT Tools (ipdSummary version 3.0). My goal is to extract ipdRatio and modified bases. In the ipdSummary --help command : ...
Marjan's user avatar
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How critical is the "Non-unique read group integer IDs found" warning in running ipdSummary?

I am trying to get the ipdRatio and modified bases information from a PacBio bam file which is sorted and indexed. The command I'm running is: ...
Marjan's user avatar
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pacbio demultiplexing issue

I was wondering if there is a way to provide pacbio lima demultiplexing only a barcode, and let lima demux sample even if only one end has the barcode, not both?
cautree's user avatar
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Finding mutations in glycosylation sites

We are going to look at the likely N- and O- glycosylation sites within MUC16 (Q8WXI7 · MUC16_HUMAN)§ by in house long-read DNA sequencing data (PacBio). Counting the number of tandem repeats is a ...
Zizogolu's user avatar
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Pacbio HIFI pbmm2 alignment metrics

I am new to pacbio sequencing data. I just did some alignment of pacbio HiFI data using pbmm2. I have the bam now, I would like to collect some metrics on the alignment. I used to work on the illumina ...
cautree's user avatar
  • 139
2 votes
1 answer
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StringTie discovers less novel isoforms when reference annotation is provided

I am analyzing PacBio IsoSeq data and I am using StringTie to assemble transcripts. I noticed the difference in the output files when I add a reference genome annotation (-G option in StringTie). In ...
Aleksandra Greshnova's user avatar
3 votes
1 answer
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regex: samtools command to "refine" PacBio IsoSeq data?

I am working with preprocessed data (IsoSeq PacBio) and I cannot understand one of the steps: samtools view -b -e [rq] >= 0.9 flnc.bam What exactly was ...
Aleksandra Greshnova's user avatar
1 vote
1 answer
59 views

How to subset an SRA file for a single chromosome?

I used prefetch to get the Pacbio reads of chicken from the SRA database. I want to align these reads against a reference genome, but not all the reads. I am only interested in a particular region on ...
venkatesh war's user avatar
3 votes
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588 views

Aligning PacBio HiFi reads to reference genome using pbmm2

I am trying to align a yeast strain sequenced by PacBio HiFi reads to the reference genome S288C (https://www.ncbi.nlm.nih.gov/data-hub/genome/GCF_000146045.2/) using pbmm2 but for some reason I am ...
rimo's user avatar
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3 votes
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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 ...
Caterina's user avatar
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1 vote
1 answer
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How can Iso-Seq reverse transcriptase artifacts be avoided?

My end goal is annotate a de-novo assembled genome. When trying to select the best method for transcriptome assembly I read Iso-seq was the preferred method. However other people suggested Nanopore as ...
Caterina's user avatar
  • 307
2 votes
2 answers
133 views

What is the most accurate approach for de Novo sequencing?

I'm trying to decide between PacBio HiFi or Illumina sequencing platforms for sequencing the genome of aChrysina scarab. We want to identify the color pattern loci and also perform a phylogenetic ...
Caterina's user avatar
  • 307
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2 answers
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Assemble Anaeroplasma species genome from metagenomic PacBio data

I have a fasta file containing reads generated by PacBio HiFi whole genome sequencing of a feces sample from mouse. I would like to use this dataset to generate an assembled circularized genome for an ...
Amroon's user avatar
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1 vote
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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 ...
Anik Dutta's user avatar
2 votes
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67 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 ...
Oakheart's user avatar
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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, ...
Anik Dutta's user avatar
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1 answer
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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 ...
Anik Dutta's user avatar
1 vote
3 answers
530 views

Polishing PacBio or ONT with Illumina

Which tool would be good to polish PacBio or ONT with Illumina? Thank you in advance
user977828's user avatar
0 votes
1 answer
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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 ...
bishopia's user avatar
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2 answers
191 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-...
ilam engl's user avatar
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1 answer
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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 ...
Jesse's user avatar
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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 ...
WilGos's user avatar
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1 vote
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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 ...
Life_Searching_Steps's user avatar
1 vote
1 answer
105 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 ...
719016's user avatar
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2 votes
0 answers
345 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 ...
Enset's user avatar
  • 21
2 votes
1 answer
68 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 ...
menten's user avatar
  • 21
4 votes
3 answers
79 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 ...
brian's user avatar
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2 votes
3 answers
254 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 ...
Biomagician's user avatar
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1 vote
1 answer
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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 ...
fridaymeetssunday's user avatar
8 votes
4 answers
11k views

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 ...
SmallChess's user avatar
  • 2,699
5 votes
1 answer
200 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....
user1238097's user avatar
4 votes
2 answers
446 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: ...
d_kennetz's user avatar
  • 631
9 votes
2 answers
320 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). ...
aechchiki's user avatar
  • 2,676
1 vote
1 answer
73 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 ...
user977828's user avatar
8 votes
2 answers
2k views

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. ...
aechchiki's user avatar
  • 2,676
6 votes
1 answer
75 views

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),...
Ian Sudbery's user avatar
  • 3,321
2 votes
1 answer
706 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 ...
Biomagician's user avatar
  • 2,459
5 votes
1 answer
106 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 ...
MayWangmeiyue's user avatar
2 votes
1 answer
286 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 ...
Biomagician's user avatar
  • 2,459
1 vote
3 answers
299 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: ...
Biomagician's user avatar
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2 votes
1 answer
192 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 ...
Biomagician's user avatar
  • 2,459
7 votes
1 answer
1k views

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: $$C = L*N / G$$...
aechchiki's user avatar
  • 2,676
10 votes
2 answers
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 ...
Mark Ebbert's user avatar
  • 1,354
11 votes
1 answer
4k views

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 ...
Mark Ebbert's user avatar
  • 1,354
6 votes
2 answers
162 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 ...
Manuel's user avatar
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