Library prep with <100ng DNA?

[sstarkenburg]

What is the lowest amount of DNA I can get away with using for a library prep for a 36bp single read run on the IIX?

[NextGenSeq]

Nextera's protocol only uses 50ng. However, the library insert size is not very tight with it. You could probably use almost any protocol if you increase the amplification cycle number. This would also increase artifacts tho.

[sstarkenburg]

Thanks for the tip. Have you had any success/experience using Illumina's library prep kit at 100ng? If so, how many amplification cycles would you recommend?

[henry.wood]

I've fairly routinely used around 50ng. I've occasionally dipped down lower. According to my picogreen machine, my lowest is 5ng. We tweaked the method a bit, doing 18 cycles of PCR before the gel cut rather than 12 cycles afterwards. It doesn't always work, and sometimes we get a bit of PCR duplicatation, but 50ng is reasonably well behaved.

[Efortier]

We routinely use 10 ng and 12 cycles without any problems. Just be careful to reduce the amount of adapters and PCR primers to take into consideration the lower amount of initial DNA.

[suludana]

Are you talking about the preparation of Genomic DNA?

[henry.wood]

I'm talking about genomic DNA, mostly for copy number purposes. I've put all the method details in a recent paper http://nar.oxfordjournals.org/cgi/co...&pmid=20525786

[sstarkenburg]

Many thanks to everyone for the input! Happy sequencing....

[sstarkenburg]

Originally Posted by Efortier

We routinely use 10 ng and 12 cycles without any problems. Just be careful to reduce the amount of adapters and PCR primers to take into consideration the lower amount of initial DNA.

How are your purifying the prep after ligating the adapters?

[mimi_lupton]

Originally Posted by henry.wood

I'm talking about genomic DNA, mostly for copy number purposes. I've put all the method details in a recent paper http://nar.oxfordjournals.org/cgi/co...&pmid=20525786

I am doing some sequencing to look at copy number variants at low depth. Can I ask why in your paper you use 200bp fragment size, did you use paired end reads? Would it have been possible to have a larger fragment size to allow you to call larger inversions for example?

Thanks

[henry.wood]

Originally Posted by mimi_lupton

I am doing some sequencing to look at copy number variants at low depth. Can I ask why in your paper you use 200bp fragment size, did you use paired end reads? Would it have been possible to have a larger fragment size to allow you to call larger inversions for example?

Thanks

Hello,
The 200bp fragment was partly due to the samples. DNA from FFPE tissues is often already fairly fragmented, so getting 500bp fragments might have been tricky. It's also partly due to convenience, that's what was already happening in the lab, so we didn't have to play around with the covaris too much.
We only used single end reads, again partly due to the fragmentation and partly because the sequencer we use mostly does single end runs. Purely from a copy number point of view, as long as you have enough sequence to trust the alignment for each read, then that's fine. To spot inversions etc using paired end reads we would have needed higher coverage. At the depth we used, we're only getting a read every 3kb or so. That's fine for copy number, and we keep the libraries, so we can always go back and resequence a sample if we want greater resolution.
Hope that makes sense.

[maggie]

I understand using less adaptor for lower input amounts during Illumina library construction and then altering the number of PCR cycles. Why/how should the PCR primer amounts be adjusted (how much should be used for 50ng input, or 200ng input, etc)?

[zwirko]

I'm trying to automate the Nextera prep, and I'm wondering how to go about the tagmentation cleanup. Yields following Ampure (SPRI) have been substantially lower than with MinElute columns, but columns can't easily be scaled up and the elution volumes on Qiagen plates are way too large not to mention highly variable. Ideally, I'd like to eliminate the tagmentation cleanup all together, but I'm not sure how since simply skipping it doesn't seem to work. Ideas?

[HoneyBee]

Originally Posted by henry.wood

Hello,
The 200bp fragment was partly due to the samples. DNA from FFPE tissues is often already fairly fragmented, so getting 500bp fragments might have been tricky. It's also partly due to convenience, that's what was already happening in the lab, so we didn't have to play around with the covaris too much.
We only used single end reads, again partly due to the fragmentation and partly because the sequencer we use mostly does single end runs. Purely from a copy number point of view, as long as you have enough sequence to trust the alignment for each read, then that's fine. To spot inversions etc using paired end reads we would have needed higher coverage. At the depth we used, we're only getting a read every 3kb or so. That's fine for copy number, and we keep the libraries, so we can always go back and resequence a sample if we want greater resolution.
Hope that makes sense.

Have you tried exome sequencing on FFPE samples? We are thinking of using TruSeq Exome kit but don't know if it works with low input + FFPE samples..

[henry.wood]

We are currently awaiting the results of FFPE exomes. We used the illumina nextera exome kits. They worked with <100ng DNA from FFPE samples. The libraries looked fine, but we won't know how the data turns out for a week or so.
I know people have used sureselect kits with FFPE in the past, but I think they need higher amounts of template. I don't know about truseq.

[HoneyBee]

Great! We did think of using Nextera exome kit but have no experience on that before. Can you let me know how the result turns out when it is out? That would be great help to decide how to proceed. Many thanks.

[kmcarr]

Originally Posted by henry.wood

We are currently awaiting the results of FFPE exomes. We used the illumina nextera exome kits. They worked with <100ng DNA from FFPE samples. The libraries looked fine, but we won't know how the data turns out for a week or so.
I know people have used sureselect kits with FFPE in the past, but I think they need higher amounts of template. I don't know about truseq.

Hey Henry,

I would like to reiterate HoneyBee's question. Were you successful using the Nextera DNA prep/exome enrichment with FFPE samples? How degraded was the DNA prior library prep and what was the final library size distribution?

Thanks.

[henry.wood]

I'd forgotten I said I'd report back. Oops. So the libraries worked OK. The DNA was reasonably good quality for FFPE, so degraded a bit, but still possible to do 500bp PCRs. It was proper surgical material pulled out of the archives though, not just quickly fixed and then immediately extracted, so a reasonable test. The libraries all had a size range between 200bp and 1000bp with normalish distribution. We did some fresh libraries at the same time and the FFPE ones looked the same.
When we did the sequencing, we were a little disappointed with the percentage on target, around 50-60, but this was the same for fixed and fresh, so just a product of the kit, rather than the samples. For what we want to do, this makes the sequencing a bit pricey, as we can restrict ourselves to only the best samples and use sureselect, so we then don't need to sequence as much to get the same depth. If sequencing cost isn't such an issue, but you only have a few samples which absolutely must get done, then it would be fine.

[HoneyBee]

wow~ Good news that Nextera exome works on FFPE samples.
May I know the fragment size of your FFPE samples? We are now working on some FFPE samples with fragment size < 100 bp... majority at ~50 bp.. How can you get FFPE DNA of good quality? We never get DNA larger than 200 bp..
Btw, just heard from Illumina that they are going to discontinue Nextera exome kit. Are you seeking alternatives right now?

Originally Posted by henry.wood

I'd forgotten I said I'd report back. Oops. So the libraries worked OK. The DNA was reasonably good quality for FFPE, so degraded a bit, but still possible to do 500bp PCRs. It was proper surgical material pulled out of the archives though, not just quickly fixed and then immediately extracted, so a reasonable test. The libraries all had a size range between 200bp and 1000bp with normalish distribution. We did some fresh libraries at the same time and the FFPE ones looked the same.
When we did the sequencing, we were a little disappointed with the percentage on target, around 50-60, but this was the same for fixed and fresh, so just a product of the kit, rather than the samples. For what we want to do, this makes the sequencing a bit pricey, as we can restrict ourselves to only the best samples and use sureselect, so we then don't need to sequence as much to get the same depth. If sequencing cost isn't such an issue, but you only have a few samples which absolutely must get done, then it would be fine.

[henry.wood]

I guess I'm a bit luckier than you with my sample sizes. Most of the FFPE samples have a fairly wide size range. The peak is around 100-200bp, but the tail often goes as high as 700bp. We prepare the DNA using the QIAamp DNA micro kit. I suspect that the kit doesn't matter too much, but that the damage gets done when the tissue is fixed. There are a wide range of fixing protocols, and some will damage the DNA more than others. We have no control over this, as we get our samples from the archives. We have plenty that don't work, but we don't attempt to sequence them.
I hadn't heard that the nextera exome kits were stopping. We only used them as a trial. We'll probably go with sure select in future, since the percentage of reads on target is better. I've heard there are some FFPE tweaks you can do with their methods, but I haven't tried them yet.