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Forum: De novo discovery 08-14-2019, 06:31 AM
Replies: 7
Views: 190
Posted By SNPsaurus
Have you thought about using PacBio for long...

Have you thought about using PacBio for long amplicons instead of tagmenting and trying to re-assemble? Each amplicon could get multiple polymerase passes for a high-quality consensus, and be...
Forum: Bioinformatics 08-04-2019, 11:24 AM
Replies: 2
Views: 192
Posted By SNPsaurus
Are these proteins DNA-binding proteins, or are...

Are these proteins DNA-binding proteins, or are you wanting to find which DNA-binding proteins bind to the gene region of these proteins?
Forum: General 08-02-2019, 03:18 PM
Replies: 6
Views: 378
Posted By SNPsaurus
Those are all super cool, but I think the root of...

Those are all super cool, but I think the root of the problem is that sequencers are multiple-fold more complex than any of the other devices. A centrifuge spins fast, PCR heats and cools, but...
Forum: Bioinformatics 07-12-2019, 01:56 PM
Replies: 10
Views: 749
Posted By SNPsaurus
Our local facility offers 2x300 v3 MiSeq runs...

Our local facility offers 2x300 v3 MiSeq runs with 25 million reads and a 2x150 v2 run with 15 million reads. If I didn't need the extra 10 million reads, I might be tempted to just do 2x150 and...
Forum: Bioinformatics 07-12-2019, 09:37 AM
Replies: 10
Views: 749
Posted By SNPsaurus
I'm curious why you would select 2x300 for a 290...

I'm curious why you would select 2x300 for a 290 bp amplicon. Did you want to merge the paired reads for extra-high quality sequence?
Forum: Sample Prep / Library Generation 06-24-2019, 12:38 PM
Replies: 4
Views: 685
Posted By SNPsaurus
If they loaded PhiX at 5% but the library was...

If they loaded PhiX at 5% but the library was mis-estimated and actually much higher, then the actual PhiX could be lower, leading to poor quality scores.

Are you sequencing just one of the cut...
Forum: Sample Prep / Library Generation 06-19-2019, 12:00 PM
Replies: 4
Views: 685
Posted By SNPsaurus
Did the facility add extra PhiX to help the...

Did the facility add extra PhiX to help the basecalling in the low-complexity region of the cut site? What is the cut site?

It does seem like two issues. If you are getting lots of off-target...
Forum: Illumina/Solexa 06-08-2019, 09:27 PM
Replies: 2
Views: 650
Posted By SNPsaurus
I think most academic cores run the cheaper high...

I think most academic cores run the cheaper high output with 8 lanes rather than the fast and expensive rapid run mode and make users wait for a run of the appropriate type to fill up. Academic users...
Forum: General 06-05-2019, 10:04 PM
Replies: 1
Views: 746
Posted By SNPsaurus
Circulomics (makers of the HMW kit) has...

Circulomics (makers of the HMW kit) has customized the protocol for a particular species and sent back DNA. You might try them, although I don't know if they rush for a time crunch.

Have you...
Forum: Vendor Forum 05-31-2019, 12:14 PM
Replies: 0
Views: 698
Posted By SNPsaurus
PacBio Sequel II services

We think the PacBio Sequel II should force a giant change in how people approach routine sequencing work. In the past, the usual progression was to do the bulk of sequencing with Illumina short...
Forum: Sample Prep / Library Generation 05-23-2019, 11:11 AM
Replies: 4
Views: 644
Posted By SNPsaurus
I see, that makes sense for amplified mtDNA. ...

I see, that makes sense for amplified mtDNA.

You can combine the Nextera Flex and Nextera XT barcode sets for Nextera Flex preps. We do that for increased multiplexing.
Forum: Sample Prep / Library Generation 05-22-2019, 12:47 PM
Replies: 4
Views: 644
Posted By SNPsaurus
Can you multiplex that much on a Miseq and get...

Can you multiplex that much on a Miseq and get enough reads per sample to reconstruct the mitogenome? I guess if the nuclear genome isn't large then a reasonable fraction of reads will go to the...
Forum: Bioinformatics 05-21-2019, 01:13 PM
Replies: 2
Views: 500
Posted By SNPsaurus
wtdbg2 will overlap long reads and then...

wtdbg2 will overlap long reads and then correct/consensus as a separate step (I know this doesn't help getting Canu to do it that way, just following up on colindaven's comment).

Canu corrects...
Forum: Sample Prep / Library Generation 05-19-2019, 09:52 PM
Replies: 5
Views: 621
Posted By SNPsaurus
It could help since a randomly biased pool is...

It could help since a randomly biased pool is unlikely to be biased over and over so you could find SNPs where the shift in allele frequencies is consistent over time. On the other hand, seeing...
Forum: Sample Prep / Library Generation 05-19-2019, 09:06 PM
Replies: 5
Views: 621
Posted By SNPsaurus
It sounds like you want to do more than SNP...

It sounds like you want to do more than SNP discovery, so my previous suggestion wouldn't be very good. But I would still suggest blasting away with whole genome sequencing for two reasons (and I say...
Forum: Sample Prep / Library Generation 05-19-2019, 08:18 PM
Replies: 5
Views: 621
Posted By SNPsaurus
If the goal is just to discover SNPs, I would...

If the goal is just to discover SNPs, I would just Nextera prep 20 individuals and sequence them to 40X read depth. Assemble the pooled reads into a draft genome, align the reads to the genome and...
Forum: Sample Prep / Library Generation 05-15-2019, 02:21 PM
Replies: 1
Views: 484
Posted By SNPsaurus
The rapture approach uses the sheared end to...

The rapture approach uses the sheared end to detect PCR clones (non-clones are unlikely to have the same break point). With ddRAD and your variant protocol you can't do that, but then you can do...
Forum: Illumina/Solexa 05-06-2019, 06:58 PM
Replies: 10
Views: 1,283
Posted By SNPsaurus
This paper has some info on phosphate's role in...

This paper has some info on phosphate's role in transposition with an indirect role: https://www.ncbi.nlm.nih.gov/pubmed/18790806
Forum: Pacific Biosciences 05-04-2019, 07:48 PM
Replies: 2
Views: 658
Posted By SNPsaurus
The source code for pbbam...

The source code for pbbam (https://github.com/PacificBiosciences/pbbam/blob/develop/src/ChemistryTable.cpp) has this table, which says that the 2.3 basecaller is for the P6-C4 chemistry.
//...
Forum: Bioinformatics 04-26-2019, 09:25 AM
Replies: 5
Views: 886
Posted By SNPsaurus
Hey, great! I was wondering if this was resolved...

Hey, great! I was wondering if this was resolved and what you said makes perfect sense and that's a good solution as well.
Forum: Illumina/Solexa 04-18-2019, 09:32 AM
Replies: 1
Views: 650
Posted By SNPsaurus
Interesting difference between NextSeq Rapid and...

Interesting difference between NextSeq Rapid and HO. The rapid groups with the examp platforms, while HO looks like the other random cluster platforms.
Forum: Illumina/Solexa 04-09-2019, 09:07 AM
Replies: 5
Views: 506
Posted By SNPsaurus
It would be helpful to say exactly what libraries...

It would be helpful to say exactly what libraries you made and then paste some example sequences of what you got back. Were most samples successfully demultiplexed? What do those reads look like?
Forum: Illumina/Solexa 04-08-2019, 09:50 PM
Replies: 5
Views: 506
Posted By SNPsaurus
It sounds to me like the reads were...

It sounds to me like the reads were demultiplexed, but you had given incorrect index sequences for a few samples, so those reads are still in the Undetermined file and you want to extract them? Is...
Forum: Bioinformatics 04-04-2019, 02:54 PM
Replies: 5
Views: 886
Posted By SNPsaurus
When I read your post I wondered if index hopping...

When I read your post I wondered if index hopping would cause the problem, and your attachment says there is an enormous level of hopping. So wouldn't samples that have a shared P1 inline barcode be...
Forum: Bioinformatics 03-20-2019, 09:23 AM
Replies: 6
Views: 649
Posted By SNPsaurus
I think luc is correct. You can inspect the bam...

I think luc is correct. You can inspect the bam file to see if the paired reads map to the same location. You can also run bbmerge and see if the two reads overlap and what percent of the library...
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