Plasmid library sequencing-which platform (if any)?

zaratieg · 01-14-2013, 11:25 AM

Hi everyone

I was hoping to pick your brains for a second. We have a plasmid library in e coli. We would like to obtain sequences form hundred of individual plasmids, starting from the backbone (meaning from an arbitrary primer) into the inserts.

The classical way to do this is miniprep and sanger sequencing of individual colonies. At the numbers that we would like to do, the price goes well into NGS territory.

I've been trying to find an NGS platform that would provide similar information from a plasmid prep of pooled colonies. But all the methods I've seen involve library prep and introducing an adapter that will serve as the primer for sequencing. Simple shotgun sequencing (by illumina for instance) of such a sample would give us the information, but only <5% of the reads would be informative (the rest would be backbone).

I just know that I haven't looked at all the miriad methods that have been developed. Is there a technology that will give you sequence from an arbitrary primer? Around 400bp would be great, but we could live with less.

Thanks in advance!

chadn737 · 01-14-2013, 11:33 AM

How big are your inserts? Because even if <5% of reads were informative, that may be enough. A single lane on a HiSeq gives well over 100 million reads anymore, so if only 5% of reads are informative, that is still several million informative reads. Even if you have a couple hundred plasmids, you will have tens of thousands of 100 bp reads covering that.

zaratieg · 01-14-2013, 03:04 PM

Thank you Chadn, that's what I was figuring. I was wondering if there was a more elegant/less brute force approach, but i guess that we are at a point where it really is economical to kill flies with a 155 howitzer.

pmiguel · 01-15-2013, 10:52 AM

You could just pool the plasmids and amplify the inserts using vector primers, then create libraries from the amplified DNA.

--
Phillip

swbarnes2 · 01-22-2013, 12:01 PM

As another poster mentioned, PCR amplify your insert, if you really want to avoid backbone. You can even put Illumina adaptors at the ends of your PCR primers, so you don't have to do any kind of library prep.

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01-14-2013, 11:25 AM	#1
zaratieg Member Location: NYC Join Date: Jun 2010 Posts: 18	Plasmid library sequencing-which platform (if any)? Hi everyone I was hoping to pick your brains for a second. We have a plasmid library in e coli. We would like to obtain sequences form hundred of individual plasmids, starting from the backbone (meaning from an arbitrary primer) into the inserts. The classical way to do this is miniprep and sanger sequencing of individual colonies. At the numbers that we would like to do, the price goes well into NGS territory. I've been trying to find an NGS platform that would provide similar information from a plasmid prep of pooled colonies. But all the methods I've seen involve library prep and introducing an adapter that will serve as the primer for sequencing. Simple shotgun sequencing (by illumina for instance) of such a sample would give us the information, but only <5% of the reads would be informative (the rest would be backbone). I just know that I haven't looked at all the miriad methods that have been developed. Is there a technology that will give you sequence from an arbitrary primer? Around 400bp would be great, but we could live with less. Thanks in advance!

01-14-2013, 11:33 AM	#2
chadn737 Senior Member Location: US Join Date: Jan 2009 Posts: 392	How big are your inserts? Because even if <5% of reads were informative, that may be enough. A single lane on a HiSeq gives well over 100 million reads anymore, so if only 5% of reads are informative, that is still several million informative reads. Even if you have a couple hundred plasmids, you will have tens of thousands of 100 bp reads covering that.

01-14-2013, 03:04 PM	#3
zaratieg Member Location: NYC Join Date: Jun 2010 Posts: 18	Thank you Chadn, that's what I was figuring. I was wondering if there was a more elegant/less brute force approach, but i guess that we are at a point where it really is economical to kill flies with a 155 howitzer.

01-15-2013, 10:52 AM	#4
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	You could just pool the plasmids and amplify the inserts using vector primers, then create libraries from the amplified DNA. -- Phillip

01-22-2013, 12:01 PM	#5
swbarnes2 Senior Member Location: San Diego Join Date: May 2008 Posts: 912	As another poster mentioned, PCR amplify your insert, if you really want to avoid backbone. You can even put Illumina adaptors at the ends of your PCR primers, so you don't have to do any kind of library prep.