454 Library / Emulsion / Instrument Protocols (October 2008)

ECO · 12-29-2008, 08:55 PM

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

DNAcowboy · 01-05-2009, 02:29 PM

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 · 03-09-2009, 10:04 AM

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 · 03-09-2009, 12:18 PM

Quote:

Originally Posted by ECO

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 · 03-24-2009, 03:51 PM

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 · 05-07-2009, 12:58 PM

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

sdstella · 05-14-2009, 03:46 PM

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 · 05-21-2009, 02:04 PM

hey Stella,

you got mail!
see you in Edinburgh!

Is Nicola already on her maternity leave?

lmsiew · 05-23-2009, 02:12 AM

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

sdstella · 05-24-2009, 01:26 PM

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

mccallumj · 06-08-2009, 02:23 PM

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 · 08-12-2009, 12:44 AM

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 · 08-12-2009, 08:25 AM

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

mccallumj · 08-12-2009, 02:19 PM

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 · 08-13-2009, 02:12 AM

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

Apollo704 · 02-01-2010, 02:18 PM

Does anyone have MVE protocols?

giakr84 · 04-08-2010, 07:04 AM

Hi all,
I need informations,
What's the maximum number of samples that I could put inside a 454 GS Junior's microtiter plate ?
Thanks

lidaof · 05-25-2010, 01:56 AM

is there any protocol for library preparation when transcriptome sequencing using 454?

thx

oyjenvol · 06-28-2010, 11:26 PM

i will use 454 for transcriptome sequencing of ploids
so if anybody have done such work please give me some suggestions
thank you very much
EMAIL: jenvol@gmail.com

LFMar · 03-01-2011, 05:59 AM

Has anyone used adaptors with expired date for 454 rapid library preparation? I've bought the adaptors (up to 10 samples) but I used them for just 2 DNA samples, now I have 8 more samples for library prep and the adaptors date just expired. I don't know if I try to use them or if I buy new ones.

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03-09-2009, 10:04 AM	#3
engencore Junior Member Location: south carolina Join Date: Mar 2009 Posts: 9	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).

03-24-2009, 03:51 PM	#5
FLXTech Member Location: UK Join Date: May 2008 Posts: 10	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.

05-07-2009, 12:58 PM	#6
lmsiew Junior Member Location: San Jose Join Date: Sep 2008 Posts: 4	454 Titanium Sequencing manual Does anyone have the latest version of 454 sequencing manual that shows the bead loading procedure? Thanks.

06-08-2009, 02:23 PM	#11
mccallumj Junior Member Location: New Zealand Join Date: May 2009 Posts: 3	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

01-05-2009, 02:29 PM	#2
DNAcowboy Member Location: france Join Date: Apr 2008 Posts: 31	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.

05-14-2009, 03:46 PM	#7
sdstella Junior Member Location: Edinburgh Join Date: May 2009 Posts: 3	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

05-21-2009, 02:04 PM	#8
Emanuel Heitlinger Member Location: Karlsruhe Join Date: Mar 2009 Posts: 11	hey Stella, you got mail! see you in Edinburgh! Is Nicola already on her maternity leave?

05-23-2009, 02:12 AM	#9
lmsiew Junior Member Location: San Jose Join Date: Sep 2008 Posts: 4	Can anyone send me a copy of the 454 Titanium Sequencing Manual please?

05-24-2009, 01:26 PM	#10
sdstella Junior Member Location: Edinburgh Join Date: May 2009 Posts: 3	I have it! Can I have your mail or something so I can send it to you?

08-12-2009, 12:44 AM	#12
jade Junior Member Location: Taiwan Join Date: Aug 2009 Posts: 2	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.

08-12-2009, 08:25 AM	#13
Bertie Junior Member Location: Pennsylvania Join Date: Jun 2009 Posts: 2	jade, take a look at this - it might help. http://gsl.cals.ncsu.edu/media/cDNA.pdf

08-12-2009, 02:19 PM	#14
mccallumj Junior Member Location: New Zealand Join Date: May 2009 Posts: 3	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.

08-13-2009, 02:12 AM	#15
jade Junior Member Location: Taiwan Join Date: Aug 2009 Posts: 2	cDNA sequencing Many thanks to Bertie and mccallumj. The info are indeed very helpful!

02-01-2010, 02:18 PM	#16
Apollo704 Junior Member Location: Boston Join Date: Jul 2009 Posts: 7	MVE Protocols? Does anyone have MVE protocols?

04-08-2010, 07:04 AM	#17
giakr84 Junior Member Location: italy Join Date: Mar 2010 Posts: 2	About number of samples Hi all, I need informations, What's the maximum number of samples that I could put inside a 454 GS Junior's microtiter plate ? Thanks

05-25-2010, 01:56 AM	#18
lidaof Junior Member Location: USA Join Date: Jun 2009 Posts: 5	is there any protocol for library preparation when transcriptome sequencing using 454? thx

06-28-2010, 11:26 PM	#19
oyjenvol Junior Member Location: China Join Date: Jun 2010 Posts: 2	i will use 454 for transcriptome sequencing of ploids so if anybody have done such work please give me some suggestions thank you very much EMAIL: jenvol@gmail.com

03-01-2011, 05:59 AM	#20
LFMar Junior Member Location: brazil Join Date: Mar 2011 Posts: 7	Expired adaptors Has anyone used adaptors with expired date for 454 rapid library preparation? I've bought the adaptors (up to 10 samples) but I used them for just 2 DNA samples, now I have 8 more samples for library prep and the adaptors date just expired. I don't know if I try to use them or if I buy new ones.