JP | JPY
-
-
-
- Challenges and Chances: A Review of the 1st Stem Cell Community Day
- Summertime, and the Livin’ Is Easy…
- Follow-on-Biologics – More than Simple Generics
- Bacteria Versus Body Cells: A 1:1 Tie
- Behind the Crime Scene: How Biological Traces Can Help to Convict Offenders
- Every 3 Seconds Someone in the World Is Affected by Alzheimer's
- HIV – It’s Still Not Under Control…
- How Many Will Be Convicted This Time?
- Malaria – the Battle is Not Lost
- Physicians on Standby: The Annual Flu Season Can Be Serious
- At the Forefront in Fighting Cancer
- Molecular Motors: Think Small and yet Smaller Again…
- Liquid Biopsy: Novel Methods May Ease Cancer Detection and Therapy
- They Are Invisible, Sneaky and Disgusting – But Today It’s Their Special Day!
- How Many Cells Are in Your Body? Probably More Than You Think!
- What You Need to Know about Antibiotic Resistance – Findings, Facts and Good Intentions
- Why Do Old Men Have Big Ears?
- The Condemned Live Longer: A Potential Paradigm Shift in Genetics
- From Research to Commerce
- Chronobiology – How the Cold Seasons Influence Our Biorhythms
- Taskforce Microbots: Targeted Treatment from Inside the Body
- Eyes on Cancer Therapy
-
-
-
-
- Challenges and Chances: A Review of the 1st Stem Cell Community Day
- Summertime, and the Livin’ Is Easy…
- Follow-on-Biologics – More than Simple Generics
- Bacteria Versus Body Cells: A 1:1 Tie
- Behind the Crime Scene: How Biological Traces Can Help to Convict Offenders
- Every 3 Seconds Someone in the World Is Affected by Alzheimer's
- HIV – It’s Still Not Under Control…
- How Many Will Be Convicted This Time?
- Malaria – the Battle is Not Lost
- Physicians on Standby: The Annual Flu Season Can Be Serious
- At the Forefront in Fighting Cancer
- Molecular Motors: Think Small and yet Smaller Again…
- Liquid Biopsy: Novel Methods May Ease Cancer Detection and Therapy
- They Are Invisible, Sneaky and Disgusting – But Today It’s Their Special Day!
- How Many Cells Are in Your Body? Probably More Than You Think!
- What You Need to Know about Antibiotic Resistance – Findings, Facts and Good Intentions
- Why Do Old Men Have Big Ears?
- The Condemned Live Longer: A Potential Paradigm Shift in Genetics
- From Research to Commerce
- Chronobiology – How the Cold Seasons Influence Our Biorhythms
- Taskforce Microbots: Targeted Treatment from Inside the Body
- Eyes on Cancer Therapy
-
JP | JPY
Reducing PCR bias for NGS library construction
Lab Academy
It has been shown that thermal cyclers with a slow ramp rate can reduce bias during PCR with GC-rich targets during NGS library preparation. But there are also options to optimize your PCR protocol on a fast ramping thermal cycler with simple elongation of certain PCR steps, or adaption of the ramping speed.
Next-generation sequencing library construction includes many steps: e.g., LD-PCR (long-distance PCR, 2 kb in length), then acoustic shearing to the appropriate size and used in a standard Illumina library preparation (involving end-repair and kination, A-tailing and adapter ligation, followed by additional amplification by PCR) [1]. Library preparations can be susceptible to bias resulting from genomes that contain unusually high or low GC content and approaches have been developed to address these situations through careful selection of polymerases for PCR amplification, thermocycling, conditions and buffers [2]. But also, the thermal cycler used influences the PCR result and improvement is possible.
Steep, fast ramp rates have been shown to be inappropriate for amplification of GC rich targets (above 80 % GC). The “steep thermoprofile does not leave sufficient time above a critical threshold temperature” so that “incomplete denaturation and poor amplification” can be the result [2]. There are multiple options to improve your PCR protocol for obtaining better results, such as addition of chemicals, change of polymerase, etc.
What to consider while NGS library preparation
In this article, we highlight three prospects related to thermal cyclers with a fast heating ramp rate to reduce PCR bias when preparing NGS libraries:
1. Optimization of the PCR protocol: Improvement includes elongation of the initial denaturation time and the denaturation time in every cycle by 6-8 times than given in the protocol [2]. But this elongates the whole PCR process and lapses the most obvious advantage of a fast ramping cycler.
2. Adapt the ramping speed: Some thermal cyclers have the option to program a desired ramping speed. Like this it is easy to soften the heating and cooling to reduce bias during PCR.
3. Play with the denaturation temperature: not only elongation of the denaturation step, but also testing different denaturation temperatures can be beneficial for adequate denaturation of GC rich targets and improve the PCR result. This can be done by either programming a gradient step in the denaturation step (if the cycler only offers 1 gradient option), or by programming a gradient in the denaturation and annealing step (if the cycler offers a 2D-gradient). The advantages of a 2D gradient are obvious. Two steps of the PCR protocol can be optimized in one run, while for a thermal cycler that offers only 1 gradient two PCR runs are necessary.
Successful preparation of your NGS library
Overall, it is possible to use a fast ramping thermal cycler for NGS PCR applications, but a slower ramp rate showed beneficial results for GC-rich targets from the start without optimization effort. When it comes to the thermal cycler itself, we recommend using a slower ramping thermal cycler such as the Mastercycler® X40 with a max. of 3.3 °C/s heating ramp rate that can still be reduced if necessary.
1. Steven R. Head,1 H. Kiyomi Komori,2 Sarah A. LaMere,2 Thomas Whisenant,2 Filip Van Nieuwerburgh,3 Daniel R. Salomon,2 and Phillip Ordoukhanian. Library construction for next-generation sequencing: Overviews and challenges. Biotechniques. 2014; 56(2):61
2. Aird D, Ross MG, Chen WS, Danielsson M, Fennell T, Russ C, Jaffe DB, Nusbaum C, Gnirke A. Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries. Genome Biol. 2011;12:R18
Steep, fast ramp rates have been shown to be inappropriate for amplification of GC rich targets (above 80 % GC). The “steep thermoprofile does not leave sufficient time above a critical threshold temperature” so that “incomplete denaturation and poor amplification” can be the result [2]. There are multiple options to improve your PCR protocol for obtaining better results, such as addition of chemicals, change of polymerase, etc.
What to consider while NGS library preparation
In this article, we highlight three prospects related to thermal cyclers with a fast heating ramp rate to reduce PCR bias when preparing NGS libraries:
1. Optimization of the PCR protocol: Improvement includes elongation of the initial denaturation time and the denaturation time in every cycle by 6-8 times than given in the protocol [2]. But this elongates the whole PCR process and lapses the most obvious advantage of a fast ramping cycler.
2. Adapt the ramping speed: Some thermal cyclers have the option to program a desired ramping speed. Like this it is easy to soften the heating and cooling to reduce bias during PCR.
3. Play with the denaturation temperature: not only elongation of the denaturation step, but also testing different denaturation temperatures can be beneficial for adequate denaturation of GC rich targets and improve the PCR result. This can be done by either programming a gradient step in the denaturation step (if the cycler only offers 1 gradient option), or by programming a gradient in the denaturation and annealing step (if the cycler offers a 2D-gradient). The advantages of a 2D gradient are obvious. Two steps of the PCR protocol can be optimized in one run, while for a thermal cycler that offers only 1 gradient two PCR runs are necessary.
Successful preparation of your NGS library
Overall, it is possible to use a fast ramping thermal cycler for NGS PCR applications, but a slower ramp rate showed beneficial results for GC-rich targets from the start without optimization effort. When it comes to the thermal cycler itself, we recommend using a slower ramping thermal cycler such as the Mastercycler® X40 with a max. of 3.3 °C/s heating ramp rate that can still be reduced if necessary.
1. Steven R. Head,1 H. Kiyomi Komori,2 Sarah A. LaMere,2 Thomas Whisenant,2 Filip Van Nieuwerburgh,3 Daniel R. Salomon,2 and Phillip Ordoukhanian. Library construction for next-generation sequencing: Overviews and challenges. Biotechniques. 2014; 56(2):61
2. Aird D, Ross MG, Chen WS, Danielsson M, Fennell T, Russ C, Jaffe DB, Nusbaum C, Gnirke A. Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries. Genome Biol. 2011;12:R18
もっと読む
表示を減らす