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Forum: Bioinformatics 09-12-2019, 08:21 AM
Replies: 5
Views: 282
Posted By SNPsaurus
So this has 2 reads of the reference, but...

So this has 2 reads of the reference, but converts to a no call? Do other loci with only reads get called? There may be a firm threshold for needing >2 reads to support a call. GQ is 0, so that means...
Forum: General 09-12-2019, 08:11 AM
Replies: 9
Views: 654
Posted By SNPsaurus
You should do the entire command as Genomax...

You should do the entire command as Genomax listed (with a change, I think, I'll mention below):

for i in *.bam; do reformat.sh in=${i}.bam out=${i}.fa; done

The first part (for i in *.bam)...
Forum: General 09-11-2019, 10:58 AM
Replies: 9
Views: 654
Posted By SNPsaurus
Can you copy what you did as a command and paste...

Can you copy what you did as a command and paste it here, and then list the directory as well and paste a few filenames as well? It looks like it didn't find any bam files. Are there files ending in...
Forum: Bioinformatics 09-11-2019, 10:53 AM
Replies: 5
Views: 282
Posted By SNPsaurus
What is the output? A vcf file will typically...

What is the output? A vcf file will typically give the genotype call and then report on read depth, allele depth, genotype probabilities and quality scores for the call, mapping, and sequences. Can...
Forum: Bioinformatics 09-10-2019, 09:56 PM
Replies: 5
Views: 282
Posted By SNPsaurus
I like breseq...

I like breseq http://barricklab.org/twiki/bin/view/Lab/ToolsBacterialGenomeResequencing because it shows the evidence for each variant call and highlights regions of no coverage as well.
Forum: Bioinformatics 09-09-2019, 08:25 PM
Replies: 4
Views: 843
Posted By SNPsaurus
That had exactly the kind of language nitpicking...

That had exactly the kind of language nitpicking I was hoping to see!
Forum: Bioinformatics 09-06-2019, 09:16 PM
Replies: 4
Views: 843
Posted By SNPsaurus
I thought these papers were really interesting...

I thought these papers were really interesting perspectives on a difficult subject. Did you get people commenting on how you could have optimized their favorite language a bit more?
Forum: De novo discovery 08-14-2019, 06:31 AM
Replies: 7
Views: 670
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: 4
Views: 649
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: 663
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: 1,058
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: 1,058
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: 851
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: 851
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: 806
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: 1,042
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: 849
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: 747
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: 747
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: 568
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: 666
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: 666
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: 666
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: 510
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: 1,336
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
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