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Forum: Bioinformatics 01-12-2015, 11:35 PM
Replies: 1
Views: 2,215
Posted By skblazer
How to read information from a kmer-distribution plot before denovo assembly

Hi guys,

I'm a dummy to this field. I know people always plot the kmer-distribution (using illumina hiseq reads or etc.) and can get a lot of information from the plot, e.g., the estimated genome...
Forum: Bioinformatics 11-27-2012, 01:21 PM
Replies: 0
Views: 963
Posted By skblazer
Consensus genotypes of SOAPsnp make no sense

Hello,

Is there anyone familiar with soapsnp? I tried soapsnp recently for SNP calling, but cannot get any SNP calls. All of the candidate variants were not called. I cannot understand why below...
Forum: Bioinformatics 08-16-2012, 10:31 AM
Replies: 0
Views: 852
Posted By skblazer
Calling SNPs and InDels simultaneously or seperately?

Hi,

I want to know when call variants by GATK pipeline, you call SNPs and InDels simultaneously (use -glm BOTH) or separately in two rounds of run. I noticed that considering BAQ will decrease the...
Forum: Sample Prep / Library Generation 11-28-2011, 12:29 PM
Replies: 12
Views: 12,438
Posted By skblazer
Many thanks again for your kindly response...

Many thanks again for your kindly response Phillip. You always answer our questions.

It seems I have to re-quantify all my DNA samples on a gel from now, or I have to purify DNA additionally using...
Forum: Sample Prep / Library Generation 11-28-2011, 07:23 AM
Replies: 12
Views: 12,438
Posted By skblazer
Problems with input DNA quantification

Dear all,

I faced the problem of quantification of input DNA for library preparation process. I attached a gel image showing six different DNA preps. According to nanodrop quantification, they...
Forum: Sample Prep / Library Generation 11-18-2011, 10:46 AM
Replies: 3
Views: 1,850
Posted By skblazer
Thanks Phillip, It's just a regular genomic...

Thanks Phillip,

It's just a regular genomic paired-end library, with PE1.0 and PE2.0 as adapters (underlined). I tried blastx, but none of them have significant hits.

I posted three sequence...
Forum: Sample Prep / Library Generation 11-18-2011, 09:14 AM
Replies: 3
Views: 1,850
Posted By skblazer
The weird insert sequence dominated my library

Hi,

I constructed an Illumina paired-end library for test, and TOPO cloned an aliquot of the library. I just received six sequence of the picked clones, all of them are adaptered fragments with...
Forum: Sample Prep / Library Generation 11-02-2011, 07:23 AM
Replies: 24
Views: 26,225
Posted By skblazer
Hi Phillip, Many thanks for the paper. ...

Hi Phillip,

Many thanks for the paper.

But do you know where the huge sodium acetate come from? Is it from buffer QG to buffer the PH? But I didn't hear a lot of people report this. I'll try...
Forum: Sample Prep / Library Generation 11-02-2011, 07:20 AM
Replies: 24
Views: 26,225
Posted By skblazer
Thanks for the advice. I'll try washing twice by...

Thanks for the advice. I'll try washing twice by PE.
Forum: Sample Prep / Library Generation 10-31-2011, 08:58 AM
Replies: 24
Views: 26,225
Posted By skblazer
I'm sorry I described inappropriately, it's not a...

I'm sorry I described inappropriately, it's not a peak at 230, it's just 260/230 is very low.
Forum: Sample Prep / Library Generation 10-31-2011, 08:57 AM
Replies: 24
Views: 26,225
Posted By skblazer
I' sorry, it's the peak shorter, it's just...

I' sorry, it's the peak shorter, it's just significantly higher at 230 than 260. (please see attached)

So the sodium acetate should not be a problem, am I right?
Forum: Sample Prep / Library Generation 10-31-2011, 07:00 AM
Replies: 24
Views: 26,225
Posted By skblazer
QIAquick Absorption Peak at 230nm rather than 260

Hello,

Each time, I used QIAquick gel extraction kit to extract the size selection product, the OD 260/230 is extremely weird (very low, close to 0), and 260/280 is a little bit higher (2.0-2.1)....
Forum: Sample Prep / Library Generation 10-27-2011, 06:15 PM
Replies: 7
Views: 5,564
Posted By skblazer
Thanks Phillip. The reaction mix is super...

Thanks Phillip. The reaction mix is super expensive, thus I think I still need this PCR purification.
Forum: Sample Prep / Library Generation 10-27-2011, 10:58 AM
Replies: 7
Views: 5,564
Posted By skblazer
All right, I'm just wondering I missed some...

All right, I'm just wondering I missed some critical steps that I don't understand.

Thanks for the advice
Forum: Sample Prep / Library Generation 10-27-2011, 10:57 AM
Replies: 7
Views: 5,564
Posted By skblazer
Sorry, I didn't mean the PCR step. I mentioned a...

Sorry, I didn't mean the PCR step. I mentioned a QIAquick PCR purification step after ligation but before size selection on a gel.

For example, the last step of "Adaptor ligation of dA-Tailed DNA"...
Forum: Sample Prep / Library Generation 10-26-2011, 05:50 PM
Replies: 12
Views: 8,401
Posted By skblazer
Many thanks, Phillip. This is very helpful.

Many thanks, Phillip. This is very helpful.
Forum: Sample Prep / Library Generation 10-26-2011, 05:50 PM
Replies: 7
Views: 5,564
Posted By skblazer
Is the PCR purification step between ligation and size selection necessary?

Hello,

This is my first time to construct an Illumina Paired-end library. According to the protocols, there is a PCR purification step after ligation of adapters and prior to size selection step....
Forum: Sample Prep / Library Generation 10-24-2011, 06:36 AM
Replies: 12
Views: 8,401
Posted By skblazer
Hi Phillip, You means it doesn't work if I...

Hi Phillip,

You means it doesn't work if I didn't anneal two oligos? But in the first reply you said I can obtain 50% recombinant moleculars of F-insert-R?

I'm sorry I'm confused with this....
Forum: Sample Prep / Library Generation 10-20-2011, 10:00 AM
Replies: 12
Views: 8,401
Posted By skblazer
Thanks Phillip, and I can't filter them out...

Thanks Phillip, and I can't filter them out through the PCR enrichment step?
Forum: Sample Prep / Library Generation 10-19-2011, 01:12 PM
Replies: 12
Views: 8,401
Posted By skblazer
Does non-Y-shaped adapter work for Illumina library?

I want to add inline barcode within custom adapters for multiplexing sequencing. The sequencing lab manager suggested me just adding the 4-6bp indexes on one end of adapter. Thus two oligos are not...
Forum: Bioinformatics 06-06-2011, 07:19 AM
Replies: 1
Views: 1,863
Posted By skblazer
Help, please......

Help, please......
Forum: Bioinformatics 06-01-2011, 09:28 AM
Replies: 1
Views: 1,863
Posted By skblazer
How to call SNPs via samtools for sex-chromosome of diploid species

Hi,

I want to call SNPs for the sex chromosomes but autosomes via samtools for one diploid individuals.

samtools mpileup -ugf ref.fa aln.bam | bcftools view -bvcg - > var.raw.bcf
bcftools...
Forum: Bioinformatics 01-12-2011, 11:59 AM
Replies: 0
Views: 2,414
Posted By skblazer
How to install ALLPATHLG?

I have used two days to try building it, but I have not fixed it.

When I tried the latest version, it failed when ./configure

g++ version is >= 4.3.3... yes
./configure: line 14507:...
Forum: Bioinformatics 01-07-2011, 04:32 PM
Replies: 8
Views: 3,750
Posted By skblazer
Thanks for your advise. I'll try.

Thanks for your advise.

I'll try.
Forum: Bioinformatics 01-07-2011, 04:30 PM
Replies: 9
Views: 3,610
Posted By skblazer
You need create a file, for example "files.txt". ...

You need create a file, for example "files.txt".
In this file, you should write the following lines:
/your/path/x.dindel_stage2_output.1.glf.txt
/your/path/x.dindel_stage2_output.2.glf.txt ...
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