454 Library / Emulsion / Instrument Protocols (October 2008)

[ECO]

Three protocol PDFs attached that I stumbled upon on the web, I thought some of you may be interested.

[DNAcowboy]

These documents are of a great help.
Do they exist for the other instruments?


C'mon Mr Illumina and Mr AB, we need some information like your friend's Mr Roche.

[engencore]

cDNA from Evrogen with Ti

Has anybody had any experience with Ti sequencing Evrogen Trimmer Direct cDNA? For a 2 region Ti, we got 67 Mb and only 20% pass. The support people feel it is a problem in the emulsion PCR. Any help would be much appreciated. We've sequenced ~30-40 similar samples with FLX and no problems (over 100 Mb, ~440,000 reads per run).

[futuro_cortina]

Hello, I´m a new member.

Three protocol PDFs attached that I stumbled upon on the web, I thought some of you may be interested.

Hello, I´m user of a 454 Flx system and I´ll use PTS (Parallel Tagged Sequence) and Selector Technology to sequencing 18 kbp of targeted genomic DNA from 500 samples, any ideas are wellcome!!
Thank you for the three protocol pdf!

[FLXTech]

Pts

Hi, hope your PTSequencing worked well. How did you sort out the data? I tried using AVA software to process my untagged reads and had problems. Any ideas appreciated.
Thanks.

[lmsiew]

454 Titanium Sequencing manual

Does anyone have the latest version of 454 sequencing manual that shows the bead loading procedure? Thanks.

[sdstella]

Hello I am a user of 454 sequencing technology. The three manuals was a treasure for me. does anyone have the GS FLX Titanium Sequencing Method Manual?Thanks

[Emanuel Heitlinger]

hey Stella,

you got mail!
see you in Edinburgh!

Is Nicola already on her maternity leave?

[lmsiew]

Can anyone send me a copy of the 454 Titanium Sequencing Manual please?

[sdstella]

I have it! Can I have your mail or something so I can send it to you?

[mccallumj]

PTS Wisdom?

Hi
We are currently working on de novo RNA-seq and re-sequencing in a pre-genome species (onion) and are interested in PTS or a similar generic barcoding approach to enable us to do population genetic studies.
If anyone can give me any opinion or advice concerning this approach I'd be really grateful. To date we are just working with barcoded primers

Regards
John

[jade]

Hi
We are trying to work out cDNA sequencing using 454, but Titanium did not work, and Standard was severely affected by polyA tail despite the nice profile of the sstDNA library.
We used Dynabeads for mRNA purification, and Stratagen Just cDNA kit for ds cDNA synthesis (1st strand primed by none-anchored oligo dT from the kit). Our samples tend to have long polyA tail, and we found 454 sequencing cannot tolerate this.
We are trying anchored dT primer now, but would appreciate any suggestion, such as choice of kit, 1st strand priming method (dT vs random primer?), or any tips in improving library constrcution for greater chance of success in sequencing?
Thank you.

[Bertie]

jade, take a look at this - it might help.
http://gsl.cals.ncsu.edu/media/cDNA.pdf

[mccallumj]

re Poly A etc

We have had very nice results from Ti sequencing of normalised cDNA prepared using Evrogen Mint + Trimmer kits, using a modified 3' Primer XXXXXXXXXXX(T)4G(T)9C(T)10VN as described by Beldade et al.

The information posted at http://www.cees.uio.no/research/faci...equencing.html was very useful.

[jade]

cDNA sequencing

Many thanks to Bertie and mccallumj. The info are indeed very helpful!