Problems with library prep from FFPE samples

FulvioChe · 04-05-2019, 04:17 AM

Hello everyone!
I'm quite new in this world, so sorry if the question is very naive.
I'm trying to do exome sequencing from FFPE samples, but the company we are working with has some troubles on obtaining a library of good values for Illumina sequencing.
The problem is that on library amplification very often the quantity of amplified library of the right size is quite low. I'm working with quite FFPE old samples (more than 30 years) and I am worried that they can be too much degraded to be sequenced; I have tried to increase the quantity of the starting materials and to increase the purity. I have to increase more this quantity/quality?
Can I give some suggestions to the company?
The kit used to extract DNA is QIAamp DNA FFPE Tissue Kit and the kit to produce the library is Agilen SureSelect V6-Post (as the company said).
Many thanks for all the help!!

nucacidhunter · 04-05-2019, 11:45 PM

If a library is prepared and includes a PCR step it should sequence well regardless of input DNA quality. Following can be useful:

1- Treating input DNA with NEB FFPE repair reagents to fix some quality aspects

2- Using a high efficiency kit such as NEBNext Ultra II or Swift Accel-NGS 1S Plus DNA Library Kit. Both will produce fully adapted libraries that may not be compatible with some SureSelect capture kits.

3- Increasing input to maximum for the library prep kit or prepping several libraries and pooling for capture

FFPE libraries also have shorter inserts so sequencing length also should be decreased to max 75 paired end or single 150 cycles if possible

These will increase the cost but more likely will result in obtaining quality data.

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04-05-2019, 04:17 AM	#1
FulvioChe Junior Member Location: Italy Join Date: Apr 2019 Posts: 1	Problems with library prep from FFPE samples Hello everyone! I'm quite new in this world, so sorry if the question is very naive. I'm trying to do exome sequencing from FFPE samples, but the company we are working with has some troubles on obtaining a library of good values for Illumina sequencing. The problem is that on library amplification very often the quantity of amplified library of the right size is quite low. I'm working with quite FFPE old samples (more than 30 years) and I am worried that they can be too much degraded to be sequenced; I have tried to increase the quantity of the starting materials and to increase the purity. I have to increase more this quantity/quality? Can I give some suggestions to the company? The kit used to extract DNA is QIAamp DNA FFPE Tissue Kit and the kit to produce the library is Agilen SureSelect V6-Post (as the company said). Many thanks for all the help!!

04-05-2019, 11:45 PM	#2
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,238	If a library is prepared and includes a PCR step it should sequence well regardless of input DNA quality. Following can be useful: 1- Treating input DNA with NEB FFPE repair reagents to fix some quality aspects 2- Using a high efficiency kit such as NEBNext Ultra II or Swift Accel-NGS 1S Plus DNA Library Kit. Both will produce fully adapted libraries that may not be compatible with some SureSelect capture kits. 3- Increasing input to maximum for the library prep kit or prepping several libraries and pooling for capture FFPE libraries also have shorter inserts so sequencing length also should be decreased to max 75 paired end or single 150 cycles if possible These will increase the cost but more likely will result in obtaining quality data.