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Forum: Pacific Biosciences 02-05-2019, 12:35 PM
Replies: 5
Views: 2,179
Posted By Magdoll
Yes both GMAP and minimap2 could work for fusion...

Yes both GMAP and minimap2 could work for fusion detection.

In GMAP it would be using the `-n 0` parameter which says "only output a single alignment *unless* the best alignment is chimeric" which...
Forum: Pacific Biosciences 02-04-2019, 07:27 PM
Replies: 5
Views: 2,179
Posted By Magdoll
BLASR is not splice aware - it cannot map...

BLASR is not splice aware - it cannot map exon-exons. Newer aligners like minimap2 are well equipped to handle errors for PacBio data. For most PacBio RNA-seq (what we call Iso-Seq) data, the error...
Forum: Pacific Biosciences 02-04-2019, 01:38 PM
Replies: 6
Views: 2,343
Posted By Magdoll
You can convert the Phred QV scores to...

You can convert the Phred QV scores to probabilities then sum over the probabilities over the entire sequence to get the expected number of errors.

You can use this Python script to calculate...
Forum: Pacific Biosciences 02-04-2019, 01:33 PM
Replies: 5
Views: 2,179
Posted By Magdoll
You cannot use BLASR. You can use minimap2, GMAP,...

You cannot use BLASR. You can use minimap2, GMAP, STAR, etc.
A tutorial for these aligners for PacBio RNA (Iso-Seq) data is available here:...
Forum: Pacific Biosciences 12-22-2017, 04:35 AM
Replies: 0
Views: 2,196
Posted By Magdoll
Register for SMRT Developers Informatics Conference at PAG 2018

Come meet the analysis experts from the PacBio community and share your bioinformatics experience!

We’re hosting a free, half-day informatics conference on Wednesday, January 17, 2018, in San...
Forum: Pacific Biosciences 10-28-2016, 09:25 AM
Replies: 1
Views: 1,987
Posted By Magdoll
Hi, Yes you are correct in your...

Hi,

Yes you are correct in your interpretation. Best way to proceed to visualizing isoforms:

-- take the HQ isoform output (polished_high_qv_consensus_isoforms.fastq) and align it to your...
Forum: Pacific Biosciences 09-06-2016, 10:09 AM
Replies: 3
Views: 1,793
Posted By Magdoll
GMAP has two parameters to filter by coverage and...

GMAP has two parameters to filter by coverage and identity. Do gmap --help for more details.

The two parameters are --min-trimmed-coverage and --min-identity.
Forum: Pacific Biosciences 08-09-2016, 02:11 PM
Replies: 3
Views: 1,224
Posted By Magdoll
Without SGE, it will be slow anyway. But if...

Without SGE, it will be slow anyway.

But if you think that single node can handle it, one way is to run multiple instances of `pbtranscript.py cluster` on the different bins.

ex: tofu_wrap.py...
Forum: Pacific Biosciences 08-08-2016, 09:29 AM
Replies: 15
Views: 3,158
Posted By Magdoll
Not sure if this problem was solved, but I almost...

Not sure if this problem was solved, but I almost wonder if some of the .bax.h5 files were actually missing (not just missing in input.fofn, but that they don't actually exist). This has occasionally...
Forum: Pacific Biosciences 08-08-2016, 09:21 AM
Replies: 3
Views: 1,793
Posted By Magdoll
Do you mean how to map PacBio transcriptome...

Do you mean how to map PacBio transcriptome (Iso-Seq) reads back to the reference genome or back to the reference transcript?

It also depends on what you have already done for the Iso-Seq data. If...
Forum: Pacific Biosciences 08-08-2016, 09:12 AM
Replies: 4
Views: 2,515
Posted By Magdoll
Hi, The simplest thing may be to run...

Hi,

The simplest thing may be to run "RS_subreads" protocol in SMRTPortal. It gives you:

# of P1 ZMWs in the "Loading" tab
Polymerase (raw) read quality in the "Filtering" tab
Mean subread...
Forum: Pacific Biosciences 08-08-2016, 09:03 AM
Replies: 3
Views: 1,224
Posted By Magdoll
Hi, Short answer is yes. There are multiple...

Hi,

Short answer is yes. There are multiple ways to hack tofu_wrap.py -- will require additional monitoring of the parallel jobs. But I first need to ask what parameters did you use to call...
Forum: Pacific Biosciences 06-17-2016, 09:56 AM
Replies: 2
Views: 2,507
Posted By Magdoll
First, our recommendation for loading for...

First, our recommendation for loading for different applications can change over time, so please always check with your local PacBio FAS or sales person to get the most up-to-date answers.
...
Forum: Pacific Biosciences 03-03-2016, 09:09 AM
Replies: 3
Views: 1,618
Posted By Magdoll
Hi, The CCS algorithm is aware of possible...

Hi,

The CCS algorithm is aware of possible incorrect adapter calling issues. It won't look at subreads that severely diverges from the rest of the subreads in terms of length and quality.

What...
Forum: Pacific Biosciences 03-01-2016, 01:44 PM
Replies: 3
Views: 1,469
Posted By Magdoll
I would like some clarification on what you mean...

I would like some clarification on what you mean by "not producing a RoI".

The Iso-Seq classify steps are:

--- using the CCS algorithm (which is generic and used for many things in addition by...
Forum: Pacific Biosciences 12-14-2015, 01:22 PM
Replies: 1
Views: 1,179
Posted By Magdoll
This paper on wheat Iso-Seq just published btw,...

This paper on wheat Iso-Seq just published btw, think you may be interested in how they did it:
http://www.biomedcentral.com/1471-2164/16/1039

They used the FLNC reads (from Iso-Seq classify) and...
Forum: Pacific Biosciences 12-02-2015, 11:08 AM
Replies: 3
Views: 1,997
Posted By Magdoll
To predict new gene functions there are a few...

To predict new gene functions there are a few ideas besides what BoHan said:

--- plain BLAST to NR to find homologs
--- PFAM or protein domain searches
(you can try using ANGEL...
Forum: Pacific Biosciences 11-09-2015, 10:03 AM
Replies: 15
Views: 2,981
Posted By Magdoll
Hi ymc, After a brief conversation with my...

Hi ymc,

After a brief conversation with my colleagues it appears that we haven't used the TruSight panel on PacBio. But we also don't see why theoretically (with some minor tweaks) it would not...
Forum: Pacific Biosciences 11-08-2015, 08:42 AM
Replies: 15
Views: 2,981
Posted By Magdoll
ymc, do you mean like capturing specific gene...

ymc, do you mean like capturing specific gene regions using using NimbleGen SeqCap EZ?
...
Forum: Pacific Biosciences 10-27-2015, 08:52 AM
Replies: 12
Views: 2,627
Posted By Magdoll
Hi, Yes the more bins the smaller each...

Hi,

Yes the more bins the smaller each individual bins will be and theoretically the faster it will be. However it also means sequences from different bins won't be clustered together. The default...
Forum: Pacific Biosciences 10-21-2015, 06:42 AM
Replies: 3
Views: 1,586
Posted By Magdoll
Hi, I don't really use Perl anymore but I...

Hi,

I don't really use Perl anymore but I think this issue is not coming from Perl but the fact that you are trying to activate a virtualenv within a script in a shell.

I found a post that...
Forum: Pacific Biosciences 10-20-2015, 12:51 PM
Replies: 11
Views: 3,257
Posted By Magdoll
1. It is better if you can access SMRTPortal at...

1. It is better if you can access SMRTPortal at least to get the CCS reads. It seems unreasonable to expect you to do all this work yet give you no computational support. Since you are not a computer...
Forum: Pacific Biosciences 10-20-2015, 10:48 AM
Replies: 1
Views: 1,801
Posted By Magdoll
Hi, I don't have personal experience with...

Hi,

I don't have personal experience with microsatellites however there seems to be some existing tools for it.

Theoretically, it should not be hard to apply most tools developed for ESTs or...
Forum: Pacific Biosciences 10-20-2015, 07:25 AM
Replies: 11
Views: 3,257
Posted By Magdoll
Hi there, First, a comprehensive resource of...

Hi there,

First, a comprehensive resource of PacBio Iso-Seq is here:
https://github.com/PacificBiosciences/cDNA_primer/wiki

The section on error correction using hybrid data is here (BUT...
Forum: Pacific Biosciences 10-02-2015, 02:10 PM
Replies: 4
Views: 2,601
Posted By Magdoll
Let me preface this by saying: what I am about to...

Let me preface this by saying: what I am about to say can be wrong (because it's been a while) and is not official.

I believe a while back we tested the TeloPrime kit in-house at PacBio. I never...
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