Directional RNA Seq

huguesparri · 02-25-2010, 08:26 AM

Hello,
I would like to know if anybody tried the "Directional RNA Seq sample prep." protocole from Illumina.
As it looks a bit like the small RNA protocole, I would like to know if people faced the same problems (adpter dimers, etc...) or if it works better.
Thanks a lot for any feedback.

Hugues

PS: I joined the protocole.

snetmcom · 02-28-2010, 09:09 PM

it's not too terrible. Data seems OK right now. It's pretty easy to avoid most of the dimers, but i would say it's impossible to avoid all.

1-2 day protocol for an average person.

huguesparri · 03-01-2010, 07:00 AM

Thanks a lot.
We're goig to try it as an alternative to DGE protocole and for bacterial RNAseq.

das · 04-13-2010, 01:04 PM

Yep. I tried it and it worked well. Let me know if you have particular questions about it.

joa_ds · 04-14-2010, 02:56 AM

I understand that the directional RNA seq on illumina is only for mRNA? Is there a total RNA protocol/kit available somewhere?

I know SOLID has a total RNA directional sequencing kit, but i don't know if that is usable on illumina, well it won't be supported anyway

Looking for directional non-coding RNA seq possibilities and only a GA2 and 454 in our facility.

das · 04-14-2010, 05:47 AM

joa_ds:
I do not think that directional protocol is limited to polyA selected RNA.
Actually directional protocol is similar to the small RNA protocol and relies on RNA ligation to preserve strand information. The issue of using polyA selected RNA has to do with with the need to get rid of abundant RNA species like ribosomal RNAs. If you do not do this, 90% of your reads will be ribosomal RNAs, which is a waste of money. One can use ribo-minus kits from Invitrogen to get rid of ribosomal RNA. From talking to other people and a little bit of my personal experience with the kit, I would suggest to do at least 2 rounds of ribo-minus depletion. However, people are discovering that even when you remove ribosomal RNA, there are several ncRNA species that are very very abundant and suck up a lot of reads. So even with ribo-minus kit, number of reads necessary to get a comprehensive coverage of the human transcriptome will be pretty high. We estimate that with polyA selected RNA-seq at 60mln reads we are getting very deep coverage of the transcriptome, so without polyA selection you probably will have generate more than 100-150 million reads to get a comprehensive transcriptome profile.
I am hoping that one day ribo-minus kit will be expanded to include probes that capture not only ribosomal RNAs but also those abundant ncRNAs.

joa_ds · 04-14-2010, 08:09 AM

thx a lot for the info ! I will let you know in some weeks if things worked out ok

Nik · 04-19-2010, 10:15 AM

Hi,

Yes, I've found this works well. However you have to steal from both sRNA and the mRNA kits as well as buying in other reagents. The key stage is the beads. Yep, 2 day protocol. Would say you start to run out of stuff pretty quick and it costs... As you use two kits you are left with bits missing and you can't do anything with. Roll on universal RNA kit illumina...

Also do any of you guys know where illumina gets the 5x frag buffer and stop solution. Will buy you a beer if so :-)

elaney_k · 04-30-2010, 09:55 AM

Quote:

Originally Posted by Nik

Hi,
Also do any of you guys know where illumina gets the 5x frag buffer and stop solution. Will buy you a beer if so :-)

I think earlier versions of the protocol used Ambions RNA Fragmentation Reagents (AM8740). It's a 10x solution so would assume you could just dilute it down... no guarantee it's still the same one provided in the kits though but worth purchasing and testing

smsm · 05-04-2010, 11:37 AM

Do you guys use AMPure beads for library purification or there are other methods. This kit is very expensive!

mx1970 · 05-18-2010, 05:12 AM

Does anybody know at which concentration is necessary adjust the small RNA sequencing primer to perform the primer hybridization for the directinal mRNA sequencing protocol?

Thanks

Nik · 05-18-2010, 06:49 AM

Hi,

Thanks elaney k.

I use AMPure beads but be aware they are bring out a new set soon if not already. Cost, well you can do a lot of libraries from 100ml and start with lower concentration samples. So initially cost is not cheap but overall 50p a sample is not bad, I’m not sure how much a column is but I think its more. And you can use robot with beads.

The RT primer is 1:5. Although I think you mean just before it goes onto the GA. I believe its set to 100uM and you don't do a diltution on the primer you get from illumina but if you buy your own, like we all do, I do the dilution to 100uM and use 6.6ul (although I use 10ul just to be sure).

link1 · 06-12-2010, 07:30 PM

Does anyone have any results or new results using this protocol...
Any other advantages of this than regular mRNA seq?

kwaraska · 07-21-2010, 08:52 AM

The original mRNA seq kit used Frag buffer and stop solution from Ambion-now AB.

The Cat# is AM8740. Just be aware that the volumes are different than the Illumina protocol. I believe you use less of the straight from Ambion kit.

som · 08-05-2010, 09:51 PM

Hi everyone,

Does anyone have problem with too small product size?
I did closely follow illumina protocol, however, the product peak size I got is around 150 nucleotides, illumina claims that the product peak should be between 200-250 nucleotides in length.

Do I need to shorten the fragmentation time (as many paired-end protocols did)? so that I could get longer fragments.
Do you think there are some problems with the adapter ligation step or else?

cmawhinney · 09-07-2010, 08:48 PM

Hi all,

I am looking at the directional protocol now and see that it requires the v1.5 sRNA 3' Adaptor. Which adaptor is this? I'm assuming it is NOT the Small RNA 3' adaptor.

Thanks!

som · 09-07-2010, 10:04 PM

v1.5 sRNA 3'adapter is a new version 1.5 small RNA 3'adapter

The v1.5 small RNA 3' adapter is specifically modified to target microRNAs
and other small RNAs that have a 3' hydroxyl group resulting from enzymatic
cleavage by Dicer or other RNA processing enzymes (illumina).

for more information about this adapter
http://seqanswers.com/forums/showthread.php?t=1265

protist · 09-08-2010, 02:44 AM

Also do any of you guys know where illumina gets the 5x frag buffer and stop solution. Will buy you a beer if so :-)[/QUOTE]

The 5X frag buffer in the published protocol is not the same as the 10X Ambion product. 10X product is a buffered Zinc solution and fragmentation is at 70C - metal based fragmentation method. The 5x solution is based on the Affy buffer (TrisAcetate + KOAc + MgOAc) and fragmentation is carried out at the higher temp of 94C.

Both buffers work well - we have not switched from the Ambion buffer and have made many RNAseq libraries both non directional and directional without issue.

cmawhinney · 09-08-2010, 09:29 AM

Quote:

Originally Posted by som

v1.5 sRNA 3'adapter is a new version 1.5 small RNA 3'adapter

The v1.5 small RNA 3' adapter is specifically modified to target microRNAs
and other small RNAs that have a 3' hydroxyl group resulting from enzymatic
cleavage by Dicer or other RNA processing enzymes (illumina).

for more information about this adapter
http://seqanswers.com/forums/showthread.php?t=1265

This is exactly what I was looking for! Thank you so much!

zorph · 09-08-2010, 10:39 AM

i just finished doing this protocol. I did directional RNA-seq prep protocol with a DSN normalization to get rid of the rRNA.
I did get a 150bp product, but illumina said it was ok.

The protocols worked well according to the bioanalyzer, but just to double check, I cloned 32 fragments into a vector prior to placing it onto the sequencing machine to see if my product and to make sure everything was ligated properly--which it was. I had 50% transcripts and 50% rRNA which, according to Illumina's recommendation, was good enough to go onto the sequencer.

Also, if someone finds fragmentation buffer, please let me know!!!!

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05-18-2010, 05:12 AM	#11
mx1970 Junior Member Location: Spain Join Date: Sep 2009 Posts: 3	RNA Sequencing primer Does anybody know at which concentration is necessary adjust the small RNA sequencing primer to perform the primer hybridization for the directinal mRNA sequencing protocol? Thanks

06-12-2010, 07:30 PM	#13
link1 Member Location: US Join Date: May 2010 Posts: 21	advantages to mRNA seq Does anyone have any results or new results using this protocol... Any other advantages of this than regular mRNA seq?

07-21-2010, 08:52 AM	#14
kwaraska Senior Member Location: Boston,MA Join Date: Nov 2008 Posts: 122	Frag buffer and stop solution The original mRNA seq kit used Frag buffer and stop solution from Ambion-now AB. The Cat# is AM8740. Just be aware that the volumes are different than the Illumina protocol. I believe you use less of the straight from Ambion kit.

08-05-2010, 09:51 PM	#15
som Junior Member Location: Tokyo Join Date: Jul 2010 Posts: 6	product peak size Hi everyone, Does anyone have problem with too small product size? I did closely follow illumina protocol, however, the product peak size I got is around 150 nucleotides, illumina claims that the product peak should be between 200-250 nucleotides in length. Do I need to shorten the fragmentation time (as many paired-end protocols did)? so that I could get longer fragments. Do you think there are some problems with the adapter ligation step or else?

02-28-2010, 09:09 PM	#2
snetmcom Senior Member Location: USA Join Date: Oct 2008 Posts: 158	it's not too terrible. Data seems OK right now. It's pretty easy to avoid most of the dimers, but i would say it's impossible to avoid all. 1-2 day protocol for an average person.

03-01-2010, 07:00 AM	#3
huguesparri Member Location: Montpellier (France) Join Date: May 2008 Posts: 93	Thanks a lot. We're goig to try it as an alternative to DGE protocole and for bacterial RNAseq.

04-13-2010, 01:04 PM	#4
das Member Location: US Join Date: Jun 2009 Posts: 16	Yep. I tried it and it worked well. Let me know if you have particular questions about it.

04-14-2010, 02:56 AM	#5
joa_ds Member Location: belgium Join Date: Dec 2008 Posts: 52	I understand that the directional RNA seq on illumina is only for mRNA? Is there a total RNA protocol/kit available somewhere? I know SOLID has a total RNA directional sequencing kit, but i don't know if that is usable on illumina, well it won't be supported anyway Looking for directional non-coding RNA seq possibilities and only a GA2 and 454 in our facility.

04-14-2010, 05:47 AM	#6
das Member Location: US Join Date: Jun 2009 Posts: 16	joa_ds: I do not think that directional protocol is limited to polyA selected RNA. Actually directional protocol is similar to the small RNA protocol and relies on RNA ligation to preserve strand information. The issue of using polyA selected RNA has to do with with the need to get rid of abundant RNA species like ribosomal RNAs. If you do not do this, 90% of your reads will be ribosomal RNAs, which is a waste of money. One can use ribo-minus kits from Invitrogen to get rid of ribosomal RNA. From talking to other people and a little bit of my personal experience with the kit, I would suggest to do at least 2 rounds of ribo-minus depletion. However, people are discovering that even when you remove ribosomal RNA, there are several ncRNA species that are very very abundant and suck up a lot of reads. So even with ribo-minus kit, number of reads necessary to get a comprehensive coverage of the human transcriptome will be pretty high. We estimate that with polyA selected RNA-seq at 60mln reads we are getting very deep coverage of the transcriptome, so without polyA selection you probably will have generate more than 100-150 million reads to get a comprehensive transcriptome profile. I am hoping that one day ribo-minus kit will be expanded to include probes that capture not only ribosomal RNAs but also those abundant ncRNAs.

04-14-2010, 08:09 AM	#7
joa_ds Member Location: belgium Join Date: Dec 2008 Posts: 52	thx a lot for the info ! I will let you know in some weeks if things worked out ok

04-19-2010, 10:15 AM	#8
Nik Member Location: London Join Date: May 2009 Posts: 14	Hi, Yes, I've found this works well. However you have to steal from both sRNA and the mRNA kits as well as buying in other reagents. The key stage is the beads. Yep, 2 day protocol. Would say you start to run out of stuff pretty quick and it costs... As you use two kits you are left with bits missing and you can't do anything with. Roll on universal RNA kit illumina... Also do any of you guys know where illumina gets the 5x frag buffer and stop solution. Will buy you a beer if so :-)

05-04-2010, 11:37 AM	#10
smsm Junior Member Location: GA Join Date: Aug 2009 Posts: 5	Do you guys use AMPure beads for library purification or there are other methods. This kit is very expensive!

05-18-2010, 06:49 AM	#12
Nik Member Location: London Join Date: May 2009 Posts: 14	Hi, Thanks elaney k. I use AMPure beads but be aware they are bring out a new set soon if not already. Cost, well you can do a lot of libraries from 100ml and start with lower concentration samples. So initially cost is not cheap but overall 50p a sample is not bad, I’m not sure how much a column is but I think its more. And you can use robot with beads. The RT primer is 1:5. Although I think you mean just before it goes onto the GA. I believe its set to 100uM and you don't do a diltution on the primer you get from illumina but if you buy your own, like we all do, I do the dilution to 100uM and use 6.6ul (although I use 10ul just to be sure).

09-07-2010, 08:48 PM	#16
cmawhinney Member Location: Boston, MA Join Date: Oct 2009 Posts: 15	Hi all, I am looking at the directional protocol now and see that it requires the v1.5 sRNA 3' Adaptor. Which adaptor is this? I'm assuming it is NOT the Small RNA 3' adaptor. Thanks!

09-07-2010, 10:04 PM	#17
som Junior Member Location: Tokyo Join Date: Jul 2010 Posts: 6	v1.5 sRNA 3'adapter is a new version 1.5 small RNA 3'adapter The v1.5 small RNA 3' adapter is specifically modified to target microRNAs and other small RNAs that have a 3' hydroxyl group resulting from enzymatic cleavage by Dicer or other RNA processing enzymes (illumina). for more information about this adapter http://seqanswers.com/forums/showthread.php?t=1265

09-08-2010, 02:44 AM	#18
protist Senior Member Location: Ireland Join Date: Jan 2009 Posts: 101	Also do any of you guys know where illumina gets the 5x frag buffer and stop solution. Will buy you a beer if so :-)[/QUOTE] The 5X frag buffer in the published protocol is not the same as the 10X Ambion product. 10X product is a buffered Zinc solution and fragmentation is at 70C - metal based fragmentation method. The 5x solution is based on the Affy buffer (TrisAcetate + KOAc + MgOAc) and fragmentation is carried out at the higher temp of 94C. Both buffers work well - we have not switched from the Ambion buffer and have made many RNAseq libraries both non directional and directional without issue.

09-08-2010, 10:39 AM	#20
zorph Member Location: FL Join Date: May 2010 Posts: 40	i just finished doing this protocol. I did directional RNA-seq prep protocol with a DSN normalization to get rid of the rRNA. I did get a 150bp product, but illumina said it was ok. The protocols worked well according to the bioanalyzer, but just to double check, I cloned 32 fragments into a vector prior to placing it onto the sequencing machine to see if my product and to make sure everything was ligated properly--which it was. I had 50% transcripts and 50% rRNA which, according to Illumina's recommendation, was good enough to go onto the sequencer. Also, if someone finds fragmentation buffer, please let me know!!!!