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Forum: RNA Sequencing 04-23-2015, 06:48 AM
Replies: 2
Views: 1,341
Posted By tokikake
Hi bastian, thank you for your fast answer! ...

Hi bastian,

thank you for your fast answer!
The rRNA/tRNA reads were filtered via mapping against the reference (and not with sortMeRNA). But the tmRNA was a good point. I added it to my...
Forum: Bioinformatics 04-23-2015, 04:40 AM
Replies: 2
Views: 3,408
Posted By tokikake
Hi Claire, if you have downloaded the...

Hi Claire,

if you have downloaded the Rfam.cm from the Rfam website (ftp://ftp.ebi.ac.uk/pub/databases/Rfam/12.0/), then you only have to run cmpress on the Rfam.cm file to prepare it for cmscan...
Forum: RNA Sequencing 04-20-2015, 02:13 AM
Replies: 2
Views: 1,341
Posted By tokikake
strange mapping bias inside outside genes in prokayotic RNASeq

Hi everyone,

as it is mentioned in the title I have encountered a strange problem in a recent RNASeq experiment which I have never seen before.

Some background:
stranded RNASeq of prokaryotic...
Forum: Bioinformatics 02-18-2015, 04:49 AM
Replies: 10
Views: 7,644
Posted By tokikake
Dear kmcarr, thanks for your reply; I have seen...

Dear kmcarr,
thanks for your reply; I have seen your answer but as they are flags for bowtie to distinguish between read orientation, I had the feeling it must be possible.

I tried tophat with...
Forum: Bioinformatics 02-13-2015, 06:43 AM
Replies: 10
Views: 7,644
Posted By tokikake
Hi, I have the same problem with bowtie2: ...

Hi,
I have the same problem with bowtie2:

I have bacterial paired-end Illumina reads from a dUTP library. bowtie2 has flags for strand specificity: "summere" only mentioned the --nofw/--norc...
Forum: RNA Sequencing 12-05-2013, 01:58 AM
Replies: 2
Views: 2,150
Posted By tokikake
Hi, I would recommend to use the ILLUMINACLIP...

Hi,
I would recommend to use the ILLUMINACLIP command; if your fragments are short you will sequence through you fragment and into the 3' adapter. It depends on your library prep and of course of...
Forum: 454 Pyrosequencing 06-28-2012, 05:10 AM
Replies: 10
Views: 2,411
Posted By tokikake
We had to pay for it but after I complained about...

We had to pay for it but after I complained about the exorbitant price (795 Euro for four tubings and five filters!!) I got one kit for free...
Forum: 454 Pyrosequencing 06-28-2012, 04:58 AM
Replies: 10
Views: 2,411
Posted By tokikake
We already replace the pump tubing/filters once....

We already replace the pump tubing/filters once. I was only irritated, that there are four (?) tubings in the kit, but you only have to replace the upper two ones. Just try it! Like GraemeFox said,...
Forum: 454 Pyrosequencing 06-18-2012, 02:06 AM
Replies: 7
Views: 2,633
Posted By tokikake
Do you see lower raw wells in your ampliocns...

Do you see lower raw wells in your ampliocns (Lib-L) as well?? Or do you sequence only Lib-A amplicons? We switched to Lib-L a few month ago; when I had a glance at our Lib-A Amplicon runs at that...
Forum: 454 Pyrosequencing 06-15-2012, 06:06 AM
Replies: 23
Views: 4,908
Posted By tokikake
we sequenced beads with an enrichment of ~30%...

we sequenced beads with an enrichment of ~30% (related to the recovered beads). When this value is reliable (and not a false good one) the sequencing results were good.
Forum: 454 Pyrosequencing 06-15-2012, 05:59 AM
Replies: 7
Views: 2,633
Posted By tokikake
lower raw wells only with RL not with Lib-L amplicons

Hi all,

we have the same experience with the low raw wells since a few month. Our lokal support also suggested the bead recovery kit as the "bad guy" and assured that 454 is working on that issue,...
Forum: Illumina/Solexa 05-21-2012, 08:46 AM
Replies: 9
Views: 2,130
Posted By tokikake
I am not sure if I get you right. Ho much is...

I am not sure if I get you right. Ho much is suitable depends on the size of your genome and the coverage you want and of course the sequencing experiment (output).
For a 2x36 bp sequencing...
Forum: Illumina/Solexa 05-21-2012, 07:31 AM
Replies: 9
Views: 2,130
Posted By tokikake
Hi vebaev, we had our GAIIx training last week...

Hi vebaev,
we had our GAIIx training last week and did the following: We prepared three additional libraries, two bacterial WGS and one cDNA library. In our training there was no time for index...
Forum: 454 Pyrosequencing 04-02-2012, 12:21 AM
Replies: 16
Views: 4,899
Posted By tokikake
As far as I know roche enlarged their sequencing...

As far as I know roche enlarged their sequencing service team, so it seems that a) they are still interested in 454 sequencing and subsequently the support of the platform or b) the problems after...
Forum: 454 Pyrosequencing 04-02-2012, 12:15 AM
Replies: 0
Views: 1,194
Posted By tokikake
Lib-L enrichment primer sequence

Hi,

as it's mentioned in the subject, I am searching for the sequence for the enrichment primer for the Lib-L Adaptors. We get often more oil from roche, so that we can do more emPCRs, but we are...
Forum: 454 Pyrosequencing 03-07-2012, 07:57 AM
Replies: 43
Views: 12,429
Posted By tokikake
I asked the same question our 1st level support:...

I asked the same question our 1st level support: We had some bad XL+ runs (WGS and 16S amplicons) and they blamed it on the length of our libraries. They said, that the new XL+ chemistry is more...
Forum: 454 Pyrosequencing 01-17-2012, 05:40 AM
Replies: 12
Views: 6,654
Posted By tokikake
We do a lot of 16S sequencing and only do...

We do a lot of 16S sequencing and only do unidirectional sequencing with the Lib-L Kit.



We also had problems after our upgrade to XL+. We compared the same libraries with Titanium and XL+...
Forum: 454 Pyrosequencing 01-17-2012, 04:52 AM
Replies: 14
Views: 10,672
Posted By tokikake
I don't know, if the sequence of the rapid...

I don't know, if the sequence of the rapid adaptors is still of interest, but here is an official document from roche.

Cheers!
Forum: 454 Pyrosequencing 12-09-2011, 07:27 AM
Replies: 19
Views: 6,502
Posted By tokikake
That is very interesting! I wrote some weeks ago...

That is very interesting! I wrote some weeks ago about our problems with the upgrade and last week we had a visit from the service and they replaced the PTP heater because of some temp problems...I...
Forum: 454 Pyrosequencing 11-22-2011, 12:15 PM
Replies: 15
Views: 4,862
Posted By tokikake
We are sequencing unidirectional amplicons and...

We are sequencing unidirectional amplicons and processing them with the shotgun pipeline. Although it is not recomended from roche to use the Lib-A Kit we don't really see a difference in the moment...
Forum: 454 Pyrosequencing 11-22-2011, 09:39 AM
Replies: 15
Views: 4,862
Posted By tokikake
Yep, I switched to the new protocols. For the...

Yep, I switched to the new protocols.
For the Amplicons we are evaluating which protocol works better, Titanium or XL+. Roche recommends to use the Titanium protocol for amplicons <650bp, but...
Forum: 454 Pyrosequencing 11-22-2011, 08:22 AM
Replies: 15
Views: 4,862
Posted By tokikake
Hi Phillip, here a some examples: XL+ ...

Hi Phillip,

here a some examples:

XL+
WGS: 64-68 Mb from a medium run; 26 Mbases from a M/S run.
Amplicons: varying between 18-27 from a M/S run and 34-52 from a medium run

FLX
WGS: 70-89...
Forum: 454 Pyrosequencing 11-22-2011, 06:56 AM
Replies: 15
Views: 4,862
Posted By tokikake
We had our upgrade some weeks ago and after the...

We had our upgrade some weeks ago and after the internal server crashed and had to be replaced, we had 3 runs on XL+ chemistry so far. Well...I am not satisfied with the performance.
With WGS...
Forum: 454 Pyrosequencing 11-22-2011, 06:21 AM
Replies: 3
Views: 2,254
Posted By tokikake
Hi, did you check, if after the second...

Hi,

did you check, if after the second melting Step the white DNA beads still cluster at the enrichment beads on the MPC?
If that is the case I would suggest to decrease the copies per bead...
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