Whether you’re getting a comprehensive view of the genome with whole genome sequencing (WGS) or focusing on the coding regions with whole exome sequencing (WES) and hybrid capture, a remaining bottleneck in next-generation sequencing (NGS) is the library preparation step. Due to risks such as sample loss and handling errors, as well as increased time and costs, optimizing this step is vital to successful experiments. This issue is magnified when working with low-input and low-quality samples.
In a recent webinar, Dr. Bhagyashree Birla from QIAGEN discussed how to work with difficult samples such as damaged DNA, single cells and ultralow-input DNA in NGS applications. After the webinar, Dr. Birla gave us the answers to some of the top questions when it comes to working with low-input and low-quality samples for whole genome sequencing.
- What is the minimum quantity or quality of DNA needed to start a WGS project?
Dr. Birla: Depending on the kit you are using, sample input can vary, but the requirement is low. A minimum of 1 ng of DNA is needed for WGS with the QIAseq FX DNA Library Kit and REPLI-g Single Cell Kit. If you’re working with ultralow amounts of DNA of around 10 pg, the QIAseq Ultralow Library Kit is the optimal choice as it ensures efficient sequencing and has one of the lowest sample-input requirements on the market. If you are working from a single cell, you can easily amplify the DNA using multiple displacement amplification with the REPLI-g Advanced Single Cell Kit.
What is the reason for lower conversion rates of ultralow sample amounts?
Dr. Birla: As DNA input amount decreases, most library kits see their conversion rates plummet and the percentage of adapter reads skyrocket. With sample amounts of 10 pg, for example, most library kits will provide around 5–10% conversion, with the majority of the reads mapped to adapter dimers. QIAGEN has addressed this issue using a combination of an end-polishing reaction, a new ligation formulation and HiFi PCR Master Mix to both convert a higher rate of DNA into libraries, as well as ensure better coverage of GC-rich regions.
Have you tested if the QIAseq Ultralow Input Library Kit is compatible with Illumina NovaSeq?
Dr. Birla: All of the QIAseq library kits are compatible with Illumina sequencers. However, they have not been tested on NovaSeq internally
Can library preparation using QIAseq library kits be automated?
Dr. Birla: Great question. Automating this step streamlines the process even further and reinforces reproducibility. QIAseq library kits are designed for automation. You can find more information on automating on Tecan, Hamilton, epMotion, and other platforms by reaching out here. The QIAseq FX DNA Library Kit has also been fully automated on epMotion 5075.
What proportion of the genome can be amplified from a single cell?
Dr. Birla: The REPLI-g Advanced DNA Single Cell Kit provides highly uniform amplification across the entire genome, with negligible sequence bias.
What are some of the advantages and limitations of using multiple displacement amplification (MDA) instead of PCR for single-cell DNA amplification?
Dr. Birla: In contrast to PCR amplification, MDA technology:
– Does not require thermal cycling, which can result in bias against high G/C sequences
– Uses Phi 29 polymerase, which has a 3’–5′ exonuclease proofreading activity to maintain 1000-fold higher fidelity during replication than Taq polymerase
– Involves fewer template binding events but very long fragments that effectively replicate the genome
– Amplifies through gDNA (or cDNA) secondary structures
How many indices can be used per sequencing run?
Dr. Birla: Currently, up to 96 indices can be used in one sequencing run.
What are the effects of using DNA with low Nanodrop 260/230 ratios when doing whole genome sequencing?
Dr. Birla: Samples with low 260/230 ratios (below about 1.8) have a significant presence of organic contaminants that may interfere with downstream library preparation steps, resulting in low or negligible yields of final library for sequencing.
What is the reason that so much DNA is lost in the final steps of a typical DNA preparation protocol?
Dr. Birla: A small amount of DNA is lost during each step of the library prep workflow, especially during bead cleanups and elution. Thus, due to decreased DNA retention at every step, the final library yield is much lower than the starting input material.
What would you recommend to improve the quality of sequencing results from damaged samples, such as FFPE, cfDNA and ChIP-seq DNA?
Dr. Birla: Using library preparation workflows that have been specifically developed and optimized for difficult samples is recommended. QIAGEN chemistries involve optimizing adapter ligation so that damaged or low-input samples are more converted into final libraries with greater efficiency.