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Forum: Sample Prep / Library Generation 05-17-2016, 11:58 PM
Replies: 4
Views: 1,164
Posted By dfhdfh
Since I had to use RiboZero anyways, I can't...

Since I had to use RiboZero anyways, I can't really see an issue with biases in rRNA depletion concerning ERCCs? Theoretically, they shouldn't bind to the probes.
The concern I have with RiboZero is...
Forum: Sample Prep / Library Generation 05-17-2016, 02:01 AM
Replies: 4
Views: 1,164
Posted By dfhdfh
Thank you for your suggestions. PolyA beads won't...

Thank you for your suggestions. PolyA beads won't be used for rRNA depletion since I have bacterial samples. RiboZero would be the way to go here. However, I am not convinced anymore if rRNA...
Forum: Sample Prep / Library Generation 05-03-2016, 07:03 AM
Replies: 4
Views: 1,164
Posted By dfhdfh
Use of ERCC spike-in for extremely different samples

Hi, I would appreciate a little help on the following situation.
I have bacterial total RNA that I separate into several samples and want to sequence afterward. From my controls I know that each of...
Forum: Illumina/Solexa 07-09-2015, 01:09 AM
Replies: 68
Views: 17,021
Posted By dfhdfh
Yeah, it's a shame.

Yeah, it's a shame.
Forum: Illumina/Solexa 06-23-2015, 06:57 AM
Replies: 29
Views: 4,526
Posted By dfhdfh
So, in my case, PhiX should cluster better than...

So, in my case, PhiX should cluster better than my library, whose size (~630 bp total) I exactly know because it's an amplicon library. However, that's not the case.
Forum: Illumina/Solexa 06-16-2015, 01:37 AM
Replies: 68
Views: 17,021
Posted By dfhdfh
After talking to Illumina, they said it might be...

After talking to Illumina, they said it might be the library but they cannot exclude a failure of the reagents, so they sent new reagents. I went down from 1000 K/mm▓ to 700 K/mm▓ and increased PhiX...
Forum: Illumina/Solexa 06-10-2015, 07:33 AM
Replies: 68
Views: 17,021
Posted By dfhdfh
Hm, that's concerning. I'm running a 2x300 v3 run...

Hm, that's concerning. I'm running a 2x300 v3 run at the moment, and now, after ~500 cycles, I have a sudden drop in quality. It's not completely finished, yet, but cycles 500-580 have a quality of...
Forum: Illumina/Solexa 06-09-2015, 01:10 AM
Replies: 29
Views: 4,526
Posted By dfhdfh
In my mind it does, because, on the one hand, I...

In my mind it does, because, on the one hand, I want as little PhiX as possible in order to maximize my actual data output. On the other hand, I want a high enough percentage in order to enable...
Forum: Illumina/Solexa 06-08-2015, 07:36 AM
Replies: 29
Views: 4,526
Posted By dfhdfh
That's a really good point. However, the...

That's a really good point. However, the libraries were all quantified in the same way and I can predict pretty well what cluster densities I'll get. So I'm actually quite confident with my...
Forum: Illumina/Solexa 06-08-2015, 06:33 AM
Replies: 29
Views: 4,526
Posted By dfhdfh
Ok, these are the results: First run (1x150...

Ok, these are the results:

First run (1x150 v3, high diversity sample), I spiked in 5 % PhiX (Illumina way), which resulted in 3.85 %. As I was quite happy with this result, I performed the second...
Forum: Illumina/Solexa 05-22-2015, 12:15 AM
Replies: 29
Views: 4,526
Posted By dfhdfh
Haha, ok :) I'll report as soon as I know...

Haha, ok :)

I'll report as soon as I know more.
Forum: Illumina/Solexa 05-21-2015, 07:34 AM
Replies: 9
Views: 1,576
Posted By dfhdfh
What is the solvent of the customer library? ...

What is the solvent of the customer library?

Edit: Buffer may be the better word, as the solvent most likely is water :D
Forum: Illumina/Solexa 05-21-2015, 07:26 AM
Replies: 29
Views: 4,526
Posted By dfhdfh
Two weeks from now, I'll run some bacterial total...

Two weeks from now, I'll run some bacterial total RNA libraries which shouldn't have too much of an issue with diversity. Maybe I'll try one the "Illumina way" and one the "pmiguel way" and see what...
Forum: Illumina/Solexa 05-21-2015, 12:03 AM
Replies: 29
Views: 4,526
Posted By dfhdfh
Well, within 1 or 2 % is just not good enough....

Well, within 1 or 2 % is just not good enough. When I'm doing amplicon sequencing and want to cut down on "wasting" reads on PhiX, I don't want to get 3 % instead of the 5 % I'm putting in. You say...
Forum: Illumina/Solexa 05-20-2015, 02:28 AM
Replies: 29
Views: 4,526
Posted By dfhdfh
Yes, this is the email I'm thinking of. I...

Yes, this is the email I'm thinking of.

I called Illumina, they said they haven't received any notifications with issues concerning these lot numbers.

I bought another new PhiX and hope it'll...
Forum: Illumina/Solexa 05-20-2015, 02:11 AM
Replies: 29
Views: 4,526
Posted By dfhdfh
Thanks for your reply. However, like I said, the...

Thanks for your reply. However, like I said, the used lot numbers were not listed by Illumina.
Forum: Illumina/Solexa 05-20-2015, 01:49 AM
Replies: 29
Views: 4,526
Posted By dfhdfh
It's not exactly on topic but close enough: ...

It's not exactly on topic but close enough:

I recently had some trouble getting my desired % of PhiX clusters. I'm doing amplicon sequencing (16 S, 2x300 v3 on MiSeq) needing higher PhiX...
Forum: Illumina/Solexa 04-24-2015, 12:51 AM
Replies: 6
Views: 3,244
Posted By dfhdfh
It's probably for the same reason the food...

It's probably for the same reason the food industry says you shouldn't refreeze thawed frozen food: When you throw it away and buy new food, they'll get more money.

In molecular biology that...
Forum: Illumina/Solexa 11-04-2014, 02:17 AM
Replies: 4
Views: 1,040
Posted By dfhdfh
I'm also planning some 16 S sequencing on a MiSeq...

I'm also planning some 16 S sequencing on a MiSeq and read that the Illumina protocol isn't the best for doing that. What do you guys recommend?
Forum: Bioinformatics 09-04-2014, 01:04 AM
Replies: 18
Views: 4,126
Posted By dfhdfh
Don't forget coolers. You really don't want your...

Don't forget coolers. You really don't want your machine to overheat.
Forum: Illumina/Solexa 07-14-2014, 07:12 AM
Replies: 5
Views: 1,166
Posted By dfhdfh
Instead of deleting, I would move them to another...

Instead of deleting, I would move them to another system or, preferably, some kind of backup system.
Forum: Illumina/Solexa 07-14-2014, 12:03 AM
Replies: 3
Views: 1,632
Posted By dfhdfh
I observed similar things as well. These runs...

I observed similar things as well. These runs more or less failed with bad clustering efficiency. Illumina support is on the case but hasn't solved anything, yet (and I don't know whether the bubbles...
Forum: Illumina/Solexa 04-25-2014, 01:33 AM
Replies: 15
Views: 8,687
Posted By dfhdfh
Regarding the gel purification: What kind of...

Regarding the gel purification: What kind of filter do you use? E.g. Illumina recommends 5 Ám filter tubes by IST Engineering. Millipore also offers 5 Ám spin filters. And then there is the classic...
Forum: Illumina/Solexa 03-12-2014, 12:58 AM
Replies: 1
Views: 1,028
Posted By dfhdfh
Are you using Bowtie 1 or 2? Bowtie 2 does it the...

Are you using Bowtie 1 or 2? Bowtie 2 does it the following way:



Source: http://bowtie-bio.sourceforge.net/bowtie2/manual.shtml


For Bowtie 1, have a look here:...
Forum: Illumina/Solexa 03-06-2014, 11:50 PM
Replies: 3
Views: 1,055
Posted By dfhdfh
With "after PCR" do you mean the PCR doesn't work...

With "after PCR" do you mean the PCR doesn't work or you lose the samples during PCR clean-up?
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