[ashchin]

Hi All,

What amount of exome do you get after 12 cycles of PCR, after hybridization using the agilent human all exon version 2.0. I have input 500ng of illumina library for capture hybridization and ended up getting around 700 ng (Based on the readings from Bioanalyzer)

PS : I have taken 28 ul of captured DNA into PCR.

Thank you,
Ashwini

[dottomarco]

I guess it is with the V2 reagents right NextGenSeq?? TrueSeq V3 should produce 375 millions paired end reads oer lane.. So now it should be possible to get 6 exomes per lane with a pretty decent coverage (75X).

Did someone try or is trying the new TrueSeq sample prep protocol that allows to skip the gel size selection? I think it was released a couple of weeks ago (regents are the same as before)

Quick summary of Illumina exome data
We get ~120 million reads per lane of HiSeq
The mean exome coverage was about 150X. Thus you can multiplex 2 to 3 whole exomes per lane.
We found our mutation so it worked pretty well.

[NextGenSeq]

We use NanoDrop and KAPA QPCR

[asiniard]

We are runnning several Illumina whole exome lanes now on the HiSeq. Once I analyze the data I'll post the coverage here.

One thing I don't like is that the protocol is very long and tedious. You do two rounds of amplification. Also, you first use the TruSeq DNA kit to make the initial library before the whole exome capture. In our hands about half the libraries didn't yield enough to continue on with the whole exome capture (you need a minimum of 500 ng).

What do you use to quantitate your libraries?

[GW_OK]

With the Truseq system we do 6 exomes per pre-capture pool, 3 lanes per pool on the Hiseq and get about 50x coverage each.

We're happy with the data so far.

[asiniard]

Thank you...very useful information. Presumably, since each kit allows up to 6 indexes one could choose 50X coverage and do 6 exomes per lane, correct?

Has anyone tried this?

[DMO]

Cluster Density

When you get 120million reads/lane, what does your cluster density look like?

Quick summary of Illumina exome data
We get ~120 million reads per lane of HiSeq
The mean exome coverage was about 150X. Thus you can multiplex 2 to 3 whole exomes per lane.
We found our mutation so it worked pretty well.

[Geneus]

Quick summary of Illumina exome data
We get ~120 million reads per lane of HiSeq
The mean exome coverage was about 150X. Thus you can multiplex 2 to 3 whole exomes per lane.
We found our mutation so it worked pretty well.

Thank you...very useful information. Presumably, since each kit allows up to 6 indexes one could choose 50X coverage and do 6 exomes per lane, correct?

[NextGenSeq]

Quick summary of Illumina exome data
We get ~120 million reads per lane of HiSeq
The mean exome coverage was about 150X. Thus you can multiplex 2 to 3 whole exomes per lane.
We found our mutation so it worked pretty well.

[starlight312]

Yes. The insert size for the HMW buffer with Nextera is much larger than the target insert of 175bp for the Agilent SureSelect protocol. This is going to affect the efficiency of the hyb and the resulting pull down. If they get it right, nextera+ Agilent targeted capture+MiSeq will be the go-to combo for Clinical Sequencing.

I'm might have this wrong and I'm not too knowledgeable when it comes to the numbers but I'd like to ask, what is the insert size range optimal for the HMW buffer for Nextera? I ask this because the current optimal insert length for the HiSeq is from 200-500bp. Assuming that the MiSeq is using identical chemistry w/ the HiSeq, then either way, we can't have the insert sizes too large (800bp+) because that's when it gets really messy.

Thanks =D

[krobison]

The 454 protocol for SureSelect shows a fragment size peak close to 700 bp; anyone know if specificity is lower with the 454 kit?

Of course, 454 has long reads -- with paired ends, you do run a bigger risk of your bait correctly capturing an exon in the middle of the fragment but your reads mostly covering neighboring DNA.

[DMO]

Yes. The insert size for the HMW buffer with Nextera is much larger than the target insert of 175bp for the Agilent SureSelect protocol. This is going to affect the efficiency of the hyb and the resulting pull down. If they get it right, nextera+ Agilent targeted capture+MiSeq will be the go-to combo for Clinical Sequencing.

[ymc]

I was told that they are targeting the clinical sequencing market. The MiSeq automates the library and cluster generation using the EpiCentre Nextera library reagents. Apparently thats why Illumina bought Epicentre.

EpiCentre says they are working on making Nextera works with Agilent SureSelect system. If they are successful in making it work, will that mean much lower DNA input, shorter prep time and no Covaris needed??

[NextGenSeq]

I was told that they are targeting the clinical sequencing market. The MiSeq automates the library and cluster generation using the EpiCentre Nextera library reagents. Apparently thats why Illumina bought Epicentre.

[ymc]

from our experience, a minimum 4-4.5 Gb needs to be collected for the Agilent exome. it does not appear that MiSeq was designed with exome resequencing in mind; but certainly a smaller targeted capture would be appropriate.

Thanks for your reply. So what is the error rate per base you can achieve with such coverage???