How fast is your NGS library preparation?

BioDynami · 08-17-2016, 08:07 PM

Everyone wants NGS library preparation to be easy, simple, and fast. The reality is that protocol times for NGS library prep kits vary from vendor to vendor. There are several factors that are closely related, such as total protocol time, hands-on time and the number of steps. The table below shows the protocol times for library preparation spanning from 50 min to several hours. The protocol times shown here start from their beginning step to the end of adaptor-ligated library purification.

It is no surprise that the protocol time is correlated with the number of steps. In addition, more steps mean longer hands-on time.

Does speed matter?

In the case of clinical sequencing, the answer is definitely yes. Speed is critical due to the nature of clinical urgency. Several clinical facilities are pushing hard to speed up their sequencing procedures, including NGS library preparation.

Moreover, time saving is not the only benefit from a faster protocol. Work becomes simpler and easier as well.

On the other hand, high speed doesn't means to sacrifice data quality or to increase cost. It’s always great when work is done in a fast, easy, and simple way with high quality and low cost.

huguesparri · 08-22-2016, 08:37 AM

Hello,

I've been looking throught the manual of your kits and in both of them, the final step is the following:
"Amplification of the DNA library created from this kit may be necessary dependent on the type of your library adaptor and sequencing platform. Prior to proceeding to PCR or other downstream steps, quantify your library to determine whether the library is enough for your application. Amplify your library according to the instruction from your platform provider."

Is this step mandatory for each library prep?
And if yes, why didn't you include it in the library prep time?

Thanks a lot for your answer,

Hugues

BioDynami · 08-22-2016, 05:48 PM

It is dependent on the adaptor you used. If you use Truseq adaptor, you can skip the PCR step. If paired end adaptor was used, then it is necessary to do the PCR. Many people like to pool samples together, they use the PCR step to add index barcodes for their samples.

ECO · 08-23-2016, 01:49 PM

Moving to Vendor Forum where this type of post belongs...

luc · 08-25-2016, 10:42 AM

Would you have some example data?
The protocol looks very similar to other single-tube library prep kits/protocols with the incubation times shortened a bit.

BioDynami · 08-25-2016, 06:54 PM

Quote:

Originally Posted by luc

Would you have some example data?
The protocol looks very similar to other single-tube library prep kits/protocols with the incubation times shortened a bit.

BioDynami NGS DNA Library Prep Kit

We have compared with other kits. Please take a look at this:

This is the library conversion efficiency comparison:

This is the coverage comparison of a difficult region for whole genome NGS: High GC + repeat sequences

DNATECH · 10-14-2016, 03:27 PM

Do the kits come with adapters? If so how many different barcodes?

BioDynami · 10-14-2016, 06:07 PM

Quote:

Originally Posted by DNATECH

Do the kits come with adapters? If so how many different barcodes?

Adaptors are not included in the kits because that will give our customers more flexibility to use different types of adaptors.

Any dA-tailed adaptor will work with our kits.

If you have multiple samples, you can construct the libraries using regular illumina paired end adaptor, then add barcode information to the libraries by PCR using index primers.

gringer · 10-16-2016, 07:55 PM

ONT 1D fragmentation kit: 15 minute sample prep, 2 mins after loading to first base-called result.

luc · 10-17-2016, 10:24 AM

... and once ONT works reliably and generates more sequence data than pain and suffering we would all like to switch.

Quote:

Originally Posted by gringer

ONT 1D fragmentation kit: 15 minute sample prep, 2 mins after loading to first base-called result.

gringer · 10-17-2016, 11:09 AM

How much sequencing data do you need? With the R9 run I did yesterday, I got 45,000 reads of ~600bp in about an hour, mean 1D accuracy of 85%, mean 2D accuracy of 91%. Bioinformatics time for preliminary amplicon read quantification (using BWA) was about half an hour.

Current (R9.4, as of today) flow cells generate 5-10Gb per run, with mean accuracy of 90-97% depending on sequencing speed and 1D/2D choice. Cost per run is still the same ($600-$1000 USD, depending on how many flow cells are purchased in bulk), which is now much cheaper than PacBio and similar to MiSeq due to increased run yield.

luc · 10-18-2016, 09:57 AM

R9.4? Please keep us updated. ONT tends to have a long lag time between their announcements and the performance delivered (years).

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08-17-2016, 08:07 PM	#1
BioDynami Registered Vendor Location: Alabama Join Date: Jul 2016 Posts: 23	How fast is your NGS library preparation? Everyone wants NGS library preparation to be easy, simple, and fast. The reality is that protocol times for NGS library prep kits vary from vendor to vendor. There are several factors that are closely related, such as total protocol time, hands-on time and the number of steps. The table below shows the protocol times for library preparation spanning from 50 min to several hours. The protocol times shown here start from their beginning step to the end of adaptor-ligated library purification. It is no surprise that the protocol time is correlated with the number of steps. In addition, more steps mean longer hands-on time. Does speed matter? In the case of clinical sequencing, the answer is definitely yes. Speed is critical due to the nature of clinical urgency. Several clinical facilities are pushing hard to speed up their sequencing procedures, including NGS library preparation. Moreover, time saving is not the only benefit from a faster protocol. Work becomes simpler and easier as well. On the other hand, high speed doesn't means to sacrifice data quality or to increase cost. It’s always great when work is done in a fast, easy, and simple way with high quality and low cost.

08-22-2016, 08:37 AM	#2
huguesparri Member Location: Montpellier (France) Join Date: May 2008 Posts: 93	Hello, I've been looking throught the manual of your kits and in both of them, the final step is the following: "Amplification of the DNA library created from this kit may be necessary dependent on the type of your library adaptor and sequencing platform. Prior to proceeding to PCR or other downstream steps, quantify your library to determine whether the library is enough for your application. Amplify your library according to the instruction from your platform provider." Is this step mandatory for each library prep? And if yes, why didn't you include it in the library prep time? Thanks a lot for your answer, Hugues

08-22-2016, 05:48 PM	#3
BioDynami Registered Vendor Location: Alabama Join Date: Jul 2016 Posts: 23	It is dependent on the adaptor you used. If you use Truseq adaptor, you can skip the PCR step. If paired end adaptor was used, then it is necessary to do the PCR. Many people like to pool samples together, they use the PCR step to add index barcodes for their samples.

08-23-2016, 01:49 PM	#4
ECO --Site Admin-- Location: SF Bay Area, CA, USA Join Date: Oct 2007 Posts: 1,358	Moving to Vendor Forum where this type of post belongs...

08-25-2016, 10:42 AM	#5
luc Senior Member Location: US Join Date: Dec 2010 Posts: 452	Would you have some example data? The protocol looks very similar to other single-tube library prep kits/protocols with the incubation times shortened a bit.

10-14-2016, 03:27 PM	#7
DNATECH Member Location: Davis, CA Join Date: Mar 2015 Posts: 29	Do the kits come with adapters? If so how many different barcodes?

10-16-2016, 07:55 PM	#9
gringer David Eccles (gringer) Location: Wellington, New Zealand Join Date: May 2011 Posts: 838	ONT 1D fragmentation kit: 15 minute sample prep, 2 mins after loading to first base-called result.

10-17-2016, 11:09 AM	#11
gringer David Eccles (gringer) Location: Wellington, New Zealand Join Date: May 2011 Posts: 838	How much sequencing data do you need? With the R9 run I did yesterday, I got 45,000 reads of ~600bp in about an hour, mean 1D accuracy of 85%, mean 2D accuracy of 91%. Bioinformatics time for preliminary amplicon read quantification (using BWA) was about half an hour. Current (R9.4, as of today) flow cells generate 5-10Gb per run, with mean accuracy of 90-97% depending on sequencing speed and 1D/2D choice. Cost per run is still the same ($600-$1000 USD, depending on how many flow cells are purchased in bulk), which is now much cheaper than PacBio and similar to MiSeq due to increased run yield.

10-18-2016, 09:57 AM	#12
luc Senior Member Location: US Join Date: Dec 2010 Posts: 452	R9.4? Please keep us updated. ONT tends to have a long lag time between their announcements and the performance delivered (years).