Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • 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!!

  • #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

    • seqadmin
      Current Approaches to Protein Sequencing
      by seqadmin


      Proteins are often described as the workhorses of the cell, and identifying their sequences is key to understanding their role in biological processes and disease. Currently, the most common technique used to determine protein sequences is mass spectrometry. While still a valuable tool, mass spectrometry faces several limitations and requires a highly experienced scientist familiar with the equipment to operate it. Additionally, other proteomic methods, like affinity assays, are constrained...
      04-04-2024, 04:25 PM
    • seqadmin
      Strategies for Sequencing Challenging Samples
      by seqadmin


      Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...
      03-22-2024, 06:39 AM

    ad_right_rmr

    Collapse

    News

    Collapse

    Topics Statistics Last Post
    Started by seqadmin, 04-11-2024, 12:08 PM
    0 responses
    18 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 04-10-2024, 10:19 PM
    0 responses
    22 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 04-10-2024, 09:21 AM
    0 responses
    17 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 04-04-2024, 09:00 AM
    0 responses
    49 views
    0 likes
    Last Post seqadmin  
    Working...
    X