Unconfigured Ad

Collapse
X
 
  • Filter
  • Time
  • Show
Clear All
new posts
  • FulvioChe
    Junior Member
    • Apr 2019
    • 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!!
  • nucacidhunter
    Jafar Jabbari
    • Jan 2013
    • 1250

    #2
    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.

    Comment

    Latest Articles

    Collapse

    • SEQadmin2
      Advanced Sequencing Platforms Tackle Neuroscience’s Toughest Genomics Problems
      by SEQadmin2



      Genomics studies in neuroscience face a special challenge due to the brain’s complexity and scarcity of samples. Mapping changes in cell type and state using conventional next-generation sequencing methods remains challenging. Advances in technologies like single-cell sequencing, spatial transcriptomics, and long-read sequencing have opened the door to deeper studies of the brain and diseases like Alzheimer’s, amyotrophic lateral sclerosis (ALS), and schizophrenia.
      ...
      07-09-2026, 11:10 AM
    • SEQadmin2
      Cancer Drug Resistance: The Lingering Barrier to Rising Survival
      by SEQadmin2



      Cancer survival rates have significantly increased in the last few decades in the United States, reaching a combined 70% 5-year survival rate by 2021. Behind this number, there are years of research to find new therapies, drug targets, and early detection methods. But there is one core challenge that keeps slowing down these advances, and it’s about drug resistance.

      There is no single reason why many patients don’t respond to treatment as expected. Cancer is...
      07-08-2026, 05:17 AM
    • GATTACAT
      Reply to Nine Things a Sample Prep Scientist Thinks About Before Sequencing
      by GATTACAT
      Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.
      07-01-2026, 11:43 AM

    ad_right_rmr

    Collapse

    News

    Collapse

    Topics Statistics Last Post
    Started by SEQadmin2, 07-13-2026, 10:26 AM
    0 responses
    22 views
    0 reactions
    Last Post SEQadmin2  
    Started by SEQadmin2, 07-09-2026, 10:04 AM
    0 responses
    32 views
    0 reactions
    Last Post SEQadmin2  
    Started by SEQadmin2, 07-08-2026, 10:08 AM
    0 responses
    20 views
    0 reactions
    Last Post SEQadmin2  
    Started by SEQadmin2, 07-07-2026, 11:05 AM
    0 responses
    34 views
    0 reactions
    Last Post SEQadmin2  
    Working...