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Forum: Illumina/Solexa 03-21-2014, 09:01 AM
Replies: 21
Views: 13,691
Posted By vtosha
May be secondary structure of DNA too difficult...

May be secondary structure of DNA too difficult for fragmentase. If 40 minutes does not help, use physical methods of fragmentation.
Forum: Illumina/Solexa 03-21-2014, 08:56 AM
Replies: 22
Views: 9,932
Posted By vtosha
You can but you should to treat them separately....

You can but you should to treat them separately. Use Qiagen kit for small RNA preparation, it help to get both types of RNA.
You can use one kit Truseq Small RNA for both types of libraries but...
Forum: Illumina/Solexa 05-23-2013, 02:52 AM
Replies: 21
Views: 13,691
Posted By vtosha
No need. I mean you should prove length just...

No need. I mean you should prove length just after treatment of fragmentase. I'm afraid, length of final libraries 250-300 too short for sequencing (length of insert 130-180). I recommend for...
Forum: Illumina/Solexa 05-23-2013, 02:40 AM
Replies: 22
Views: 9,932
Posted By vtosha
1-20 nM. We use a half dose of reagents:).

1-20 nM. We use a half dose of reagents:).
Forum: Illumina/Solexa 05-23-2013, 02:25 AM
Replies: 21
Views: 13,691
Posted By vtosha
We found no biases or difference between...

We found no biases or difference between libraries after Covaris and fragmentase. If you have no possibility to use Covaris, fragmentase - best and cheapest variant. But I advise to prove length of...
Forum: Illumina/Solexa 09-20-2012, 09:04 AM
Replies: 22
Views: 9,932
Posted By vtosha
NEB kit use heat inactivation of ligase (see...

NEB kit use heat inactivation of ligase (see instruction to ligase). As I think Illumina use NEB-reagents for it's sample prep kits, so I try to change Illumina manual and use temperature...
Forum: Sample Prep / Library Generation 09-20-2012, 08:58 AM
Replies: 8
Views: 3,386
Posted By vtosha
I got smallRNA with Qiagen Kit. Do you mean % of...

I got smallRNA with Qiagen Kit. Do you mean % of miRNA in final reads?
Forum: Sample Prep / Library Generation 09-17-2012, 03:10 AM
Replies: 8
Views: 3,386
Posted By vtosha
I think no difference which kit (Illumina or NEB)...

I think no difference which kit (Illumina or NEB) you use because Illumina get their reagents from NEB (I think so by some reason). We try both kits, on smallRNA of plant, human and fly.
Main...
Forum: Illumina/Solexa 09-17-2012, 03:05 AM
Replies: 22
Views: 9,932
Posted By vtosha
I think when small RNA is used instead of total...

I think when small RNA is used instead of total RNA then reagents are used more economically and with more good. And clear bands at the final step too.
Forum: Bioinformatics 09-16-2012, 01:25 AM
Replies: 11
Views: 2,480
Posted By vtosha
I made a thread in Russian-language forum...

I made a thread in Russian-language forum (http://molbiol.ru/forums/index.php?showtopic=515179&st=0&p=1348106) about microRNA, may be it will be useful for russian-speaking users.
Forum: Sample Prep / Library Generation 09-16-2012, 01:18 AM
Replies: 8
Views: 3,386
Posted By vtosha
We use only smallRNA as input not total RNA, for...

We use only smallRNA as input not total RNA, for plant as for animal. With total RNA as input output libraries too low.
Adapters attaching may be not so good.
And after gel cutting: faint - how...
Forum: Bioinformatics 09-07-2012, 05:23 AM
Replies: 11
Views: 2,480
Posted By vtosha
Если вопрос в том, может ли быть так, что среди...

Если вопрос в том, может ли быть так, что среди растительных микроРНК встречаются животные, то, по-моему, может (http://www.plantcell.org/content/18/12/3355.short). Более свежих примеров навскидку не...
Forum: Bioinformatics 09-07-2012, 04:49 AM
Replies: 11
Views: 2,480
Posted By vtosha
Попробуйте для обработки miRDeep2, хорошая...

Попробуйте для обработки miRDeep2, хорошая програмка. Ей можно скормить в качестве референсного любой близкий полный геном или даже просто имеющийся набор генов, она проведёт ряд фильтраций хитов по...
Forum: Illumina/Solexa 09-07-2012, 02:28 AM
Replies: 35
Views: 46,752
Posted By vtosha
We have done directional libraries from bacterial...

We have done directional libraries from bacterial mRNA using Truseq small RNA. If you have interested yet I can tell about them.
Forum: Illumina/Solexa 05-31-2012, 07:03 AM
Replies: 73
Views: 351,188
Posted By vtosha
See Bentley at al., 2008...

See Bentley at al., 2008 (http://www.ncbi.nlm.nih.gov/pubmed?term=Accurate%20whole%20human%20genome%20sequencing%20using%20reversible%20terminator%20chemistry).
Supplementary gives many valuable...
Forum: Illumina/Solexa 05-31-2012, 06:37 AM
Replies: 73
Views: 351,188
Posted By vtosha
Yes, you are right. Adaptors bind to oligos on...

Yes, you are right. Adaptors bind to oligos on glass of flowcell. These oligos are absent on picture of step 2.
Forum: Bioinformatics 03-02-2012, 02:28 AM
Replies: 27
Views: 5,634
Posted By vtosha
What software do you use for trimming and sliding...

What software do you use for trimming and sliding window?
Forum: Bioinformatics 02-29-2012, 04:16 AM
Replies: 27
Views: 5,634
Posted By vtosha
Some bacterial, near 5,5 M. 72nt, paired-end. N50...

Some bacterial, near 5,5 M. 72nt, paired-end. N50 from 100 000 to 170 000. Not the best at all, but best our. But for some chloroplast genome we couldn't get any long contig with an excellent...
Forum: Bioinformatics 02-29-2012, 04:07 AM
Replies: 27
Views: 5,634
Posted By vtosha
We try with Perl-script of our bioinformatician....

We try with Perl-script of our bioinformatician. As I understand, for IT it is not too difficult to write such script for autoscreening Velvet's parametres.
Forum: Sample Prep / Library Generation 02-29-2012, 04:03 AM
Replies: 3
Views: 1,950
Posted By vtosha
Do you measure concentration of your samples with...

Do you measure concentration of your samples with another technics? Bioanalyzer not reliable for measuring of concentration.
Forum: Bioinformatics 02-29-2012, 03:52 AM
Replies: 27
Views: 5,634
Posted By vtosha
Try k-mers more than 31. We got a good results...

Try k-mers more than 31. We got a good results from 37 to 47. -shortPaired. -exp_cov auto Try maximum number of reads your computer can treat. We got best results on 15+15 mln and 20+20 mln, less...
Forum: Illumina/Solexa 12-22-2011, 02:05 AM
Replies: 73
Views: 351,188
Posted By vtosha
Bam-format:...

Bam-format: http://bioinformatics.oxfordjournals.org/content/26/5/676.full
Forum: Illumina/Solexa 12-21-2011, 03:08 AM
Replies: 19
Views: 11,425
Posted By vtosha
Our training teacher insisted on that Qubit data...

Our training teacher insisted on that Qubit data more valid.
Forum: Illumina/Solexa 12-20-2011, 11:21 PM
Replies: 19
Views: 11,425
Posted By vtosha
Yes, it is a problem with defining of...

Yes, it is a problem with defining of concentration. I used 16 pM (Qubit) on all lanes and some lanes gave a 1M, phiX gave a 350k, and library on self-made adapters gave 400 K.
Real-time gave a...
Forum: Illumina/Solexa 12-20-2011, 03:03 AM
Replies: 19
Views: 11,425
Posted By vtosha
We got on GAIIx 1M/mm2, after filtration 600 K....

We got on GAIIx 1M/mm2, after filtration 600 K. Intensity was detected. Techsupport said about lane overload, but we got good results when assembly have done.
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