Identifying the best PCR enzyme for library amplification in NGS
April 2024
Authors:
Michael A. Quail, Craig Corton, James Uphill, Jacqueline Keane and Yong Gu
Abstract:
“PCR amplification is a necessary step in many next-generation sequencing (NGS) library preparation methods. Whilst many PCR enzymes are developed to amplify single targets efficiently, accurately and with specificity, few are developed to meet the challenges imposed by NGS PCR, namely unbiased amplification of a wide range of different sizes and GC content. As a result PCR amplification during NGS library prep often results in bias toward GC neutral and smaller fragments. As NGS has matured, optimized NGS library prep kits and polymerase formulations have emerged and in this study we have tested a wide selection of available enzymes for both short-read Illumina library preparation and long fragment amplification ahead of long-read sequencing.
We tested over 20 different hi-fidelity PCR enzymes/NGS amplification mixes on a range of Illumina library templates of varying GC content and composition, and find that both yield and genome coverage uniformity characteristics of the commercially available enzymes varied dramatically. Three enzymes Quantabio RepliQa Hifi Toughmix, Watchmaker Library Amplification Hot Start Master Mix (2X) ‘Equinox’ and Takara Ex Premier were found to give a consistent performance, over all genomes, that mirrored closely that observed for PCR-free datasets. We also test a range of enzymes for long-read sequencing by amplifying size fractionated S. cerevisiae DNA of average size 21.6 and 13.4 kb, respectively.
The enzymes of choice for short-read (Illumina) library fragment amplification are Quantabio RepliQa Hifi Toughmix, Watchmaker Library Amplification Hot Start Master Mix (2X) ‘Equinox’ and Takara Ex Premier, with RepliQa also being the best performing enzyme from the enzymes tested for long fragment amplification prior to long-read sequencing. “
Sage Science Products:
BluePippin and SageELF were used to fractionate DNA for testing long-range PCR enzymes.
Methods Excerpt:
“Sheared S. cerevisiae DNA was size fractionated using Sage Sciences ELF or Bluepippin instruments yielding modal fragment sizes of 21.6 and 13.3 kb, respectively. After adapter ligation 1 ng of each of these were used as a template for long range PCR with a range of enzymes using manufacturers recommended cycling conditions…”
Microbial Genomics
Whole genome sequencing identifies associations for nonsyndromic sagittal craniosynostosis with the intergenic region of BMP2 and noncoding RNA gene LINC01428
April 2024
Authors:
Anthony M. Musolf, Cristina M. Justice, Zeynep Erdogan-Yildirim, Seppe Goovaerts, Araceli Cuellar, John R. Shaffer, Mary L. Marazita, Peter Claes, Seth M. Weinberg, Jae Li, Craig Senders, Marike Zwienenberg, Emil Simeonov, Radka Kaneva, Tony Roscioli, Lorena Di Pietro, Marta Barba, Wanda Lattanzi, Michael L. Cunningham, Paul A. Romitti & Simeon A. Boyadjiev
Abstract:
“Craniosynostosis (CS) is a major birth defect resulting from premature fusion of cranial sutures. Nonsyndromic CS occurs more frequently than syndromic CS, with sagittal nonsyndromic craniosynostosis (sNCS) presenting as the most common CS phenotype. Previous genome-wide association and targeted sequencing analyses of sNCS have identified multiple associated loci, with the strongest association on chromosome 20. Herein, we report the first whole-genome sequencing study of sNCS using 63 proband-parent trios. Sequencing data for these trios were analyzed using the transmission disequilibrium test (TDT) and rare variant TDT (rvTDT) to identify high-risk rare gene variants. Sequencing data were also examined for copy number variants (CNVs) and de novo variants. TDT analysis identified a highly significant locus at 20p12.3, localized to the intergenic region between BMP2 and the noncoding RNA gene LINC01428. Three variants (rs6054763, rs6054764, rs932517) were identified as potential causal variants due to their probability of being transcription factor binding sites, deleterious combined annotation dependent depletion scores, and high minor allele enrichment in probands. Morphometric analysis of cranial vault shape in an unaffected cohort validated the effect of these three single nucleotide variants (SNVs) on dolichocephaly. No genome-wide significant rare variants, de novo loci, or CNVs were identified. Future efforts to identify risk variants for sNCS should include sequencing of larger and more diverse population samples and increased omics analyses, such as RNA-seq and ATAC-seq. “
Sage Science Products:
PippinHT was used to size select whole genome libraries for Oxford Nanopore Promethion sequencing.
Methods Excerpt:
“…3–5 µg of genomic DNA was sheared using a Megaruptor 3 (Diagenode) and purified using Ampure XP beads. Sheared DNA was size selected using the PippinHT instrument (Sage Science) with a target range of 16–20 kb fragments. Next, 1 µg of fragmented, purified, and size-selected DNA in a volume of 47 µl was used in the SQK-LSK109 library preparation protocol per manufacturer’s instructions (Oxford Nanopore Technologies). DNA was end-repaired using the NEBNext FFPE DNA Repair Mix and NEBNext Ultra II End Repair/dA-tailing modules, followed by purification with AMPure XP beads (1:1 vol ratio) and elution to a final volume of 60 µl. Adapters were ligated, and the final library resuspended in Long Fragment Buffer (Oxford Nanopore Technologies). The resulting final library yield was 1.2–2.2 µg per specimen. Libraries were loaded onto PromethION Flowcells (R9.4.1) with 20 femtomolar (fM) loading. After 24 h, all specimens were nuclease washed and reloaded with 20 fM of library. Total sequencing run time was 72 h.”
Nature Scientific Reports
DOI:10.1038/s41598-024-58343-w
Mature microRNA-binding protein QKI promotes microRNA-mediated gene silencing
February 2024
Authors:
Kyung-Won Min, Myung Hyun Jo, Minseok Song, Ji Won Lee, Min Ji Shim, Kyungmin Kim, Hyun Bong Park,Shinwon Ha, Hyejin Mun, Ahsan Polash, Markus Hafner, Jung-Hyun Cho, Dongsan Kim, Ji-Hoon Jeong, Seungbeom Ko, Sungchul Hohng, Sung-Ung Kang & Je-Hyun Yoon
Abstract:
“Although Argonaute (AGO) proteins have been the focus of microRNA (miRNA) studies, we observed AGO-free mature miRNAs directly interacting with RNA-binding proteins, implying the sophisticated nature of fine-tuning gene regulation by miRNAs. To investigate microRNA-binding proteins (miRBPs) globally, we analyzed PAR-CLIP data sets to identify RBP quaking (QKI) as a novel miRBP for let-7b. Potential existence of AGO-free miRNAs were further verified by measuring miRNA levels in genetically engineered AGO-depleted human and mouse cells. We have shown that QKI regulates miRNA-mediated gene silencing at multiple steps, and collectively serves as an auxiliary factor empowering AGO2/let-7b-mediated gene silencing. Depletion of QKI decreases interaction of AGO2 with let-7b and target mRNA, consequently controlling target mRNA decay. This finding indicates that QKI is a complementary factor in miRNA-mediated mRNA decay. QKI, however, also suppresses the dissociation of let-7b from AGO2, and slows the assembly of AGO2/miRNA/target mRNA complexes at the single-molecule level. We also revealed that QKI overexpression suppresses cMYC expression at post-transcriptional level, and decreases proliferation and migration of HeLa cells, demonstrating that QKI is a tumour suppressor gene by in part augmenting let-7b activity. Our data show that QKI is a new type of RBP implicated in the versatile regulation of miRNA-mediated gene silencing.“
Sage Science Products:
PippinHT was used to isolate micro RNA libraries.
Methods Excerpt:
“Library preparation was performed using the TruSeq Small RNA library preparation kit (Illumina, RS-200) following the manufacturer’s instructions. Briefly, 1 μg of input RNA was loaded into Urea-TBE gels for purification and the resulting RNAs were applied for the following procedures. User-supplied reagents including T4 RNA ligase2 Deletion Mutant (Lucigen, LR2D1132K) and Maxima First Stand cDNA synthesis kit (Thermo Fisher Scientific, K1641) were purchased separately. Libraries were amplified using 11 cycles of PCR for the manufacture’s index or modified index primer set to increase diversity. Libraries were prepared according to the manufacturer’s protocol with a modification in the size selection step, which, instead of agarose gel purification, PippinHT Prep instrument (Sage Science, HTP0001) and 3% agarose dye-free cassette with internal standards (Sage Science, HTG3010) was used under the following conditions: base pair start = 120 bp, base pair end = 160 bp, range = broad, target peak size = 145 bp. Eluted Libraries from PippinHT system were subsequently analysed on Tape-station 4150 (Agilent Technologies, G2992AA) following the manufacturer’s instructions using a High Sensitivity DNA Screen tape (Agilent Technologies, 5067–5584). Each library was barcoded with unique sequence of reverse primers during the PCR step, which contained Illumina compatible indices and modified indexes (see GEO database). Before pooling libraries for the next-generation sequencing, concentration of each library was measured in a high sensitivity Tape-station followed by smear analysis. Illumina NextSeq 550 or Miniseq with single end (50 nt;R1) read method was apply for the library sequencing.”
RNA Biology
DOI: 10.1080/15476286.2024.2314846
In vitro size-selection of short circulating tumor DNA fragments from late-stage lung cancer patients enhance the detection of mutations and aneuploidies
January 2024
Authors:
Christoffer Trier Maansson, Louise Skov Thomsen, Laura Stokkebro, Julie Gabe Dissing, Maiken Parm Ulhoi, Anders Lade Nielsen, Peter Meldgaard, Boe Sandahl Sorensen
Abstract:
“Introduction
Recent studies have demonstrated differences between the fragment length profiles of cell-free DNA (cfDNA) from cancer patients and healthy individuals. This has led to the development of in vitro size-selection procedures which can isolate the short fragments that are enriched with mutated circulating tumor DNA (ctDNA). This has yet to be investigated in a large cohort of lung cancer patients.
Materials and methods
We used plasma samples from 35 stage III and IV lung cancer patients and performed targeted next-generation sequencing (NGS) and variant calling from cfDNA with and without size-selection of short fragments. We identified clonal hematopoiesis (CH) and germline mutations using targeted NGS on paired buffy coat (BC) samples. In addition, we performed a genome-wide copy-number alteration analysis on the cfDNA samples with and without size-selection.
Results
ctDNA containing tumor mutations had a different fragment length profile compared to cfDNA fragments with CH or germline mutations. In vitro size-selection resulted in a median 1.36-fold (interquartile range (IQR): 0.63 to 2.48) mutational allele fraction (MAF) enrichment of tumor mutations whereas CH/germline mutations had a median 0.95-fold (IQR: 0.62 to 1.05) MAF enrichment. Key oncogenic drivers, including KRAS and EGFR were more likely to have a MAF increase with size-selection. Size-selection also increased the number plasma aneuploidy positive samples from 8 of 35 to 20 of 35.
Conclusion
This study expands the knowledge regarding ctDNA fragmentation in lung cancer patients and we demonstrate that in vitro size-selection can increase MAF of tumor mutations and plasma aneuploidy calls. Size-selection could lead to increased sensitivity of ctDNA detection, which is crucial for clinical implementation of liquid biopsies. This study is the largest of its kind studying cfDNA samples from 35 lung cancer patients containing 109 mutations in total.. “
Sage Science Products:
PippinHT was used to size select cfDNA samples.
Methods Excerpt:
“In vitro size-selection of cfDNA was performed using PippinHT (Sage Science, Beverly, MA, USA) according to the manufacturer’s instructions. For each sample, 20 μl of purified cfDNA was added to a 3 % agarose gel cassette. The range mode was used to collect fragments with 95–230 bp with a pause at 152 bp. This resulted in a collection of two 30 μl cfDNA fractions and the fraction containing the short fragments (95–152 bp) was subjected to quality control and CAPP-seq..”
The Journal of Liquid Biopsy
DOI: 10.1016/j.jlb.2024.100141
The haplotype-resolved T2T carnation (Dianthus caryophyllus) genome reveal the correlation between genome architecture and gene expression
November 2023
Authors:
Lan Lan, Luhong Leng, Weichao, Yonglin Ren, Wayne Reeve, Xiaopeng Fu, Zhiqiang Wu, Xiaoni Zhang
Abstract:
“Carnation (Dianthus caryophyllus) is one of the most valuable commercial 45 flowers, due to its richness of colour and form, and its excellent storage and vase life. 46 The diverse demands of the market require faster breeding in carnations. A full 47 understanding of carnations is therefore required to guide the direction of breeding. 48 Hence, we assembled the haplotype-resolved gap-free carnation genome of a variety 49 ‘Baltico’ which is the most common white standard variety worldwide. Based on the 50 high-depth HiFi, ultra-long nanopore and Hi-C sequencing data, we assembled the 51 telomere-to-telomere (T2T) genomes to be 564,479,117 and 568,266,215 bp, for the 52 two haplotypes Hap1 and Hap2, respectively. This T2T genome exhibited great 53 improvement in genome assembly and annotation results compared with the former 54 version. The improvements were seen when different approaches to evaluation were 55 used. Our T2T genome first informs the analysis of the telomere and centromere 56 region, enabling us to speculate about the specific centromere characteristics that 57 cannot be identified by high order repeats in carnations. We analyzed the allele-58 specific expression in three tissues and the relationship between the genome 59 architecture and gene expression in the haplotypes. This demonstrated that the length 60 of the genes, CDS, introns, the exon numbers and the transposable elements insertions 61 correlate with gene expression ratios and levels. The insertions of transposable 62 elements repress expression in gene regulatory networks in carnation. This gap-free 63 finished T2T carnation genome provides a valuable resource to illustrate the genome 64 characteristics and functional genomics analysis for further studies and molecular 65 breeding. “
Sage Science Products:
The SageHLS instrument was used to size select Ultra High Molecular Weight DNA for Oxford Nanopore Promethion sequencing (Genome Center of Grandomics, Wuhan China)
Author Affiliations:
Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Chinese Academy of Agricultural Sciences, Shenzhen, China
College of Science, Health, Engineering and Education, Murdoch University, Western Australia
Kunpeng Institute of Modern Agriculture at Foshan, Shenzhen Branch, Chinese Academy of Agricultural Sciences, Shenzhen, China
Key Laboratory of Horticultural Plant Biology, College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan, China
Preprint – Horticulture Research
DOI: 10.1093/hr/uhad244