xGen™ Predesigned NGS Amplicon Panels

Ready-to-use panels designed for research on COVID-19, oncology, inherited diseases, metagenomics, or sample ID that allow you to investigate answers to your questions.

xGen NGS—ready to react.

Árajánlatot, információt kérek!

IDT termékek szállítási költségei 2024.04.-től:

A szintézis helyétől, valamint a szállítási körülményektől függően az alábbi költségek kerülnek felszámításra:

Belgiumból: nettó 6 900 Ft
USA-ból: nettó 34 000 Ft
Belgiumból, szárazjégen: nettó 37 000 Ft

USA-ból, szárazjégen: nettó 45 000 Ft
Oldatok szállítási költsége: mennyiség és súly függvénye, kérjen ajánlatot a Bio-Science Kft.-től!

Általános tájékoztató az IDT USA gyártási központjából érkező „custom” termékcsoportokról:

szintetikus biológiai termékek:

Gének
gBlocks, eBlocks
Ultramerek
RNS-oligók
CRISPR guide RNS-ek
oPools

qPCR próbák:

a szintézis helye a szekvencia komplexitásától, valamint a választott festék/ quencher kombinációtól függ

szolgáltatások:

PAGE tisztított termékek

stock termékek:

nem áll teljes lista rendelkezésre, kérjen ajánlatot a Bio-Science Kft.-től!

xGen™ Oncology & Inherited Diseases Amplicon Panels

Compatible with FFPE and cell-free DNA samples

Confident variant identification as low as 1% allele frequency facilitates research on tumor growth and identification of driver mutations in a fast, easy, single-tube workflow.

xGen NGS—made for oncology and inherited disease research.

xGen™ Metagenomics Amplicon Panels

Research identities in mixed microbial community

16S v2 and ITS1 panels facilitate NGS research on complex microbial communities using a single primer pool targeting the 16S rRNA gene or ITS1 region. These panels can be customized with additional targets including antibiotic resistance or virulence genes.

xGen NGS—made for metagenomics.

xGen™ SARS-CoV-2 Amplicon Panels

Targeted sequencing to research SARS-CoV-2 viral genomics

Overlapping primers produce 99.7%† genomic coverage of SARS-CoV-2 lineages. These consistent primers obtain sequence data from viral concentrations as low as 10–100 viral copies without the need to evaluate new primer sets for emerging lineages.

xGen NGS—made for COVID-19 research.

xGen™ Monkeypox Virus Amplicon Panel

xGen Amplicon Technology uses overlapping primer sets to provide comprehensive coverage of positions 6760–190,905 (ITRs not included) from as few as 300 monkeypox viral genome copies. Track the spread in a single-tube, ~2.5-hour workflow.

xGen NGS—made for monkeypox research.

xGen™ Sample Identification (ID) Amplicon Panel

Track and manage samples, including matched pairs

The power of discrimination of this amplicon panel is over 1 in 85,000, making it suitable for longitudinal research studies and scenarios in which genetic fingerprinting is relevant to research design and analysis.

xGen NGS—made for sample identification research.

ARTIC SARS-CoV-2 Amplicon Panel

Multiplex PCR panel for SARS-CoV-2 amplicon sequencing

The ARTIC panel consists of two primer pools that amplify SARS-CoV-2 viral genomes from complex samples for research. The sequencing data can elucidate the presence of mutations consistent with known variants or identify new variants in research studies.

xGen NGS—made for COVID-19 research.

xGen™ SARS-CoV-2 Midnight Amplicon Panel

Multiplex PCR primers for amplicon sequencing

Panel for amplification of SARS-CoV-2 from research samples that can be used to understand the spread of the virus, identify variants, and helps enable a better understanding of COVID-19.

xGen™ NGS—made for COVID-19 research.

xGen™ HS EGFR Pathway Amplicon Panel

Targeted sequencing panel for EGFR, BRAF, KRAS, and NRAS

xGen HS EGFR Pathway Amplicon Panel can identify variants with a MAF of ≤1% from ≥ 10 ng of cell-free DNA (cfDNA) or FFPE DNA for Illumina® sequencers.

xGen NGS—made sensitive.

For research use only. Not for use in diagnostic procedures. Unless otherwise agreed to in writing, IDT does not intend for these products to be used in clinical applications and does not warrant their fitness or suitability for any clinical diagnostic use. Purchaser is solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations.

99.7% SARS-CoV-2 genomic coverage

The xGen SARS-CoV-2 Amplicon Panel uses overlapping primers to generate 345 amplicons, sized 116–255 bp (average 150 bp), along the length of the 29.9 kb viral genome, obtaining 99.7% coverage of the genome (Figure 2). Overlapping primers ensure that variants are identified, even when the mutation interferes with a primer binding site. Comprehensive mutation identification is crucial for tracking nucleotide variants and improve understanding of virus evolution, transmission, and pathogenesis.

Figure 2. The xGen SARS-CoV-2 Amplicon Panel provides 99.7% coverage of the SARS-CoV-2 genome. RNA was isolated from gamma-irradiated and sonicated cell lysate from Vero E6 cultured monkey kidney epithelial cells (ATCC® CRL-1586™) infected with SARS-CoV-2 isolate USA-WA1/2020 (BEI Resources, Cat. No. NR-52287). 100,000 copies of SARS-CoV-2 RNA was converted into cDNA using the SuperScript™ IV Kit (Thermo Fisher Scientific). The resulting NGS library generated with the xGen SARS-CoV-2 Amplicon Panel was sequenced 2 x 150 bp on a MiniSeq® System (Illumina). Resulting reads were downsampled to 280k reads per sample (n = 2) for analysis. Genomic coverage for a representative sample is shown.

Full genomic coverage of lineages with a single primer set

Using a single, consistent primer set, xGen SARS-CoV-2 Amplicon Panel has been shown to cover multiple lineages, including sub-lineages of Omicron (Figure 3), Delta, and Alpha (Figure 4). The primer set has remained unchanged since its release in 2020, preventing the need for frequent changes to primer designs.

Figure 3. Two representative examples of sequence coverage obtained from Omicron SARS-CoV-2 RNA. RNA was extracted from nasopharyngeal swabs and converted into cDNA using the SuperScript IV Kit (Thermo Fisher Scientific). The resulting library generated with the xGen SARS-CoV-2 Amplicon Panel was sequenced 2 x 150 bp on a MiniSeq System (Illumina). Resulting reads were downsampled to 83,000 reads per sample for analysis. Genomic coverage for two BA.1 lineages is shown. Ct values are 13.6 and 19.9. A total of 93 biological research samples that exhibited S gene dropout by qPCR were analyzed, 90 of which were identified as Omicron variants.

Figure 4. An example of coverages obtained from B.1.1.7 (Alpha), B.1.2, B.1.1.64, B.1.298, B.1.429 (Epsilon), B.1.617.2 (Delta), and AY.3 (Delta) SARS-CoV-2. RNA was extracted from nasopharyngeal or oropharyngeal swabs and converted into cDNA using the SuperScript IV Kit (Thermo Fisher Scientific). The resulting library generated with the xGen SARS-CoV-2 Amplicon Panel was sequenced 2 x 150 bp on a MiniSeq System (Illumina). Resulting reads were downsampled to 83,000 reads per sample for analysis. Genomic coverage for a representative sample per lineage is shown.

Research Investigation

Case Study I Genemarkers, LLC

Genemarkers, LLC, and Swift Biosciences collaborated to identify mutations and variants from SARS-CoV-2 samples sourced in Kalamazoo, Michigan. Samples were anticipated to be U.K. variants (B.1.1.7) due to the observation of S gene dropout in PCR testing. After generating an NGS library with the xGen SARS-CoV-2 Amplicon Panel and sequencing, the mutation pattern of SARS-CoV-2 B.1.1.7 was not observed in one sample, leading to clarification on variants present in the Kalamazoo area.

Obtain genomes from viral titers as low as 10–100 viral copies

10 to 1 million viral genome copies (Figure 5) are sufficient to generate NGS libraries using the xGen SARS-CoV-2 Amplicon Panel. Mixed RNA samples were converted into first strand cDNA and used to create sequencing libraries with the xGen SARS-CoV-2 Amplicon Panel. Libraries were enzymatically normalized to 4 nM using the Normalase workflow.

Figure 5. Obtain genomes from as few as 10–100 viral copies. SARS-CoV-2 RNA USA-WA1/2020 (BEI Resources, catalog # NR-52287) was mixed with Universal Human Reference RNA (Agilent, catalog # 740000) and converted into first-strand cDNA using the Superscript® IV First-Strand Synthesis System (Thermo Fisher Scientific). The resulting library generated with the xGen SARS-CoV-2 Amplicon Panel was sequenced on a MiniSeq System (Illumina) at 2 x 150 bp. Resulting reads were downsampled to 280k reads per sample (n = 2). Genomic copies identified are shown.

The recent emergence of monkeypox viral infections globally has resulted in an increased need for rapid, reliable NGS approaches to not only monitor and trace outbreaks but to also track any potentially novel variants that may arise. Epidemiological studies are currently underway to pinpoint transmission and infection patterns of this zoonotic disease. IDT recognizes the importance of these studies and has designed an xGen NGS Amplicon Sequencing Panel to target the monkeypox virus†.

The xGen Monkeypox Virus Amplicon Panel offers a streamlined (DNA-to-sequencer in 2.5 hours), single-tube NGS workflow for studying the monkeypox virus (MPXV). This Predesigned xGen Amplicon Panel provides 184 kb of high-quality coverage of the monkeypox genome (Table 2, Figure 2 and Figure 3) from inputs as low as 300 viral genome copies (Table 3). xGen Amplicon technology includes amplicon tiling and creation of super amplicons to ensure comprehensive genome coverage and provide resistance to future viral mutations that may fall on a priming site (Figure 4), thus enabling future identification of novel variants.

Coverage of the monkeypox virus genome. As an example of comprehensive coverage, at the specified positions, of monkeypox (DQ011157), ~3000 copies of the monkeypox genome (BEI Resources, NIAID, NIH: Genomic DNA from Monkeypox Virus, USA-2003, NR-4928) and 10 ng Coriell DNA NA12878 (human) were input into the xGen Amplicon workflow using the xGen Monkeypox Virus Amplicon Panel. The resulting NGS library was sequenced on a MiSeq system (Illumina) with 250 bp paired-end (PE) sequencing with 3,055,632 total reads. Reads were aligned and mapped to the monkeypox reference genome (DQ011157 [2]) using bwa (v 2.2.1 [3]). The total sequencing depth per genomic position (in log ₁₀ scale) was then plotted to visualize genomic coverage. Example data for one replicate are shown here, though experimental duplicates showed similar results."> Coverage of the monkeypox virus genome.

Figure 2. Coverage of the monkeypox virus genome. As an example of comprehensive coverage, at the specified positions, of monkeypox (DQ011157), ~3000 copies of the monkeypox genome (BEI Resources, NIAID, NIH: Genomic DNA from Monkeypox Virus, USA-2003, NR-4928) and 10 ng Coriell DNA NA12878 (human) were input into the xGen Amplicon workflow using the xGen Monkeypox Virus Amplicon Panel. The resulting NGS library was sequenced on a MiSeq system (Illumina) with 250 bp paired-end (PE) sequencing with 3,055,632 total reads. Reads were aligned and mapped to the monkeypox reference genome (DQ011157 [2]) using bwa (v 2.2.1 [3]). The total sequencing depth per genomic position (in log10 scale) was then plotted to visualize genomic coverage. Example data for one replicate are shown here, though experimental duplicates showed similar results.

Coverage and on-target mapping rates

Based on initial research and development, this panel has been shown to offer comprehensive coverage of the monkeypox viral genome from positions 6760–190,905. The inverted terminal repeats (ITRs) at both ends of the genome were omitted from the panel design due to the repetitive nature of these sequences.

To prepare amplicon sequencing libraries using the xGen Monkeypox Virus Amplicon Panel, ~3000 copies of the monkeypox genome (BEI Resources) and 10 ng Coriell DNA NA12878 (human) were used. The resulting NGS library was sequenced on a MiniSeq™ system (Illumina) with 150 bp paired-end (PE) sequencing with 1,774,058 total reads. Reads were aligned and mapped to the monkeypox reference genome (DQ011157 [2]) using bwa (v 2.2.1 [3]). Table 2 shows representative metrics obtained in this proof-of-concept experiment.

Table 2. xGen Monkeypox Virus Amplicon Panel NGS metrics.

	% mapping	% on-target (base)	% base uniformity (>0.2X mean)
xGen Monkeypox Virus Amplicon Panel	88.1	97.7	98.0

The xGen Monkeypox Virus Amplicon Panel results in comprehensive coverage of the monkeypox genome (excluding the ITRs). To prepare amplicon sequencing libraries using the xGen Monkeypox Virus Amplicon Panel, ~3000 copies of the monkeypox genome (BEI Resources, NIAID, NIH: Genomic DNA from Monkeypox Virus, USA-2003, NR-4928) and 10 ng Coriell DNA NA12878 (human) were used. The resulting NGS library was sequenced on a MiniSeq system (Illumina) with 150 bp paired-end (PE) sequencing with 1,774,058 total reads. Reads were aligned and mapped to the monkeypox reference genome (DQ011157 [2]) using bwa (v 2.2.1 [3]). The shown dot plot indicates the relative coverage of each amplicon in the panel (n = 4), where each amplicon is represented by a blue dot. Colored dashed lines represent mean coverage of 0.05X, 0.1X, 0.2X, 1X, and 5X."> The xGen Monkeypox Virus Amplicon Panel results in comprehensive coverage of the monkeypox genome.

Figure 3. The xGen Monkeypox Virus Amplicon Panel results in comprehensive coverage of the monkeypox genome (excluding the ITRs). To prepare amplicon sequencing libraries using the xGen Monkeypox Virus Amplicon Panel, ~3000 copies of the monkeypox genome (BEI Resources, NIAID, NIH: Genomic DNA from Monkeypox Virus, USA-2003, NR-4928) and 10 ng Coriell DNA NA12878 (human) were used. The resulting NGS library was sequenced on a MiniSeq system (Illumina) with 150 bp paired-end (PE) sequencing with 1,774,058 total reads. Reads were aligned and mapped to the monkeypox reference genome (DQ011157 [2]) using bwa (v 2.2.1 [3]). The shown dot plot indicates the relative coverage of each amplicon in the panel (n = 4), where each amplicon is represented by a blue dot. Colored dashed lines represent mean coverage of 0.05X, 0.1X, 0.2X, 1X, and 5X.

Sequencing results from titers as low as 300 viral genome copies

The input into xGen Monkeypox Virus Amplicon Panel consisted of either 3000, 300, or 0 copies of the monkeypox genome (BEI Resources) and 10 ng Coriell DNA NA12878 (human). The resulting NGS library was sequenced as described above. Reads were aligned and mapped to the monkeypox reference genome (DQ011157 [2]) using bwa (v 2.2.1 [3]). A high level of genomic coverage was observed with 3000 and 300 monkeypox genomic copies (Table 3), and no genomic coverage was observed with no monkeypox copy input.

Table 3. The xGen Monkeypox Virus Amplicon Panel provides coverage for a range of viral DNA inputs.

Sample number	Input copies of viral genomes	Total reads	Percent target bases 10X (base)
1	3000	2,374,500	99.4
2	3000	2,725,540	99.4
3	300	1,984,616	98.8
4	300	1,963,414	98.7
5	0/NTC	2,183,412	0.2
6	0/NTC	2,464,906	0.3

Super amplicons help maintain sequencing coverage despite mutations

The xGen Monkeypox Virus Amplicon Panel maintains genomic coverage despite mutations at primer binding sites. The generation of super amplicons means that even in the case of a mutation occurring in primer binding sites (shown here by black arrows), the xGen Monkeypox Virus Amplicon Panel can maintain genomic coverage. The input into library prep consisted of ~3000 copies of the monkeypox genome (BEI Resources) and 10 ng Coriell DNA NA12878 (human). The resulting NGS library was sequenced on a MiniSeq system (Illumina) (150 bp PE sequencing) with 1,774,058 total reads. Reads were aligned and mapped to the monkeypox reference genome (DQ011157 [2]) using bwa (v 2.2.1 [3]) and coverage was visualized with IGV (Broad Institute [4])."> The xGen Monkeypox Virus Amplicon Panel maintains genomic coverage despite mutations at primer binding sites.

Figure 4. The xGen Monkeypox Virus Amplicon Panel maintains genomic coverage despite mutations at primer binding sites. The generation of super amplicons means that even in the case of a mutation occurring in primer binding sites (shown here by black arrows), the xGen Monkeypox Virus Amplicon Panel can maintain genomic coverage. The input into library prep consisted of ~3000 copies of the monkeypox genome (BEI Resources) and 10 ng Coriell DNA NA12878 (human). The resulting NGS library was sequenced on a MiniSeq system (Illumina) (150 bp PE sequencing) with 1,774,058 total reads. Reads were aligned and mapped to the monkeypox reference genome (DQ011157 [2]) using bwa (v 2.2.1 [3]) and coverage was visualized with IGV (Broad Institute [4]).

Features	xGen 16S v2 and ITS1 Amplicon Panels
Panel information	23 primers (16S v2); average 425 bp amplicon size 15 primers (ITS1); amplicon size 145–695 bp
Input material	10 pg for microbial isolates; 1–50 ng for metagenomic samples
Time	2 hours cDNA-to-library or 3 hours cDNA-to-normalized-library-pool
Components available	Target-specific multiplex primer pool PCR and library prep reagents Normalase technology (optional, included) Dual-indexed primers Note: Kits do not include RT module or magnetic beads
Multiplexing capability	Up to 96 combinatorial dual index (CDI) or 1536 unique dual index (UDI)
Recommended depth	16S v2: 100K reads per library ITS1: 25K reads per library

Coverage of all variable regions of the 16S rRNA gene and ITS1 region

The 16S v2 and xGen ITS1 Amplicon Panels facilitate NGS analysis of complex microbial communities (e.g., bacteria, archaea, fungi) using primer pools that target the 16S rRNA gene (variable regions 1–9) and ITS1 region (Figure 2). In addition, these panels can be customized with additional targets, including antibiotic resistance or virulence genes, allowing sub-genera level identification and functional analysis.

xGen 16S Amplicon Panel v2 (primers)

xGen 16S rRNA Amplicon Panel covers all nine variable regions for NGS.

Figure 2. xGen 16S Amplicon Panel v2 (primers). Sequencing read coverage for an E. coli DNA sample (n = 1) observed in Integrative Genomics Viewer (IGV) Sashimi plot and illustration of multiplexed primer coverage of all nine variable regions of 16S rRNA compared to a standard V3/V4 16S sequencing read coverage.

xGen ITS1 Amplicon Panel (primers)

The xGen ITS1 Amplicon Panel targets the internal transcribed spacer regions of the rRNA genes

Figure 3. xGen ITS1 Amplicon Panel (primers). Sequencing read coverage observed in the IGV Sashimi plot and illustration of multiplexed primer coverage of ITS1 region for Candida albicans (n = 1). Reads originating from the forward primer are shown in red; reads from the reverse primer are in blue.

The xGen 16S Amplicon Panel v2 and the xGen ITS1 Amplicon Panel provide representation of diverse microbial communities

Comparison of 16S rRNA V3–V4 coverage to xGen 16S rRNA Amplicon Panel v2 that covers V1–V9.

Figure 4. The xGen 16S Amplicon Panel v2 provides superior representation of diverse microbial communities. The 16S v2 panel covering V1–V9 regions of 16S rRNA provides accurate representation of each genus in a commercially available standard (MSA-1003) compared to libraries interrogating the V3–V4 region alone. The legacy Accel-Amplicon 16S+ITS panel and the new xGen 16S Amplicon Panel v2 show similar relative abundance results. Strains were present at levels from 0.02% to 18% in MSA-1003. Boxed organisms were not detected with sole use of the V3-V4 region. Libraries were sequenced on a MiSeq® (Illumina) instrument without the use of the PhiX reagent. As shown in the examples in this figure, by not losing reads to PhiX or having to sequence deeper due to the use of phased primers, an increased number of samples can be fit on a sequencing run, thereby increasing sequencing efficiency, while still achieving quality data.

Comparison of two commercial microbial community standards for ITS1 coverage.

Figure 5. Expected vs. observed Zymo I/II data from the panel covering the ITS1 region. The xGen ITS1 Amplicon Panel provides accurate representation of each genus in two commercially available standards [Zymo I—ZymoBIOMICS™ Microbial Community standard and Zymo II—ZymoBIOMICS Microbial Community standard II (Log distribution)]. Fungal strains were present at levels from 0.1% to 99.9% in Zymo II. The xGen ITS1 Amplicon Panel resulted in expected representation of the fungal species in a bacterial background.

Consistent function with varying biomass, sample types, and read length

Consistent NGS data for samples of different biomass, sample type, and read length.

Figure 6. Consistent function with varying biomass, sample type, and read length. Using the same protocol and cycling conditions, input quantities of 10 ng to 50 ng of swine manure (top left) identified the same quantities and types of genera. In addition, the 10 pg sample of MSA-1003 (bottom left) had the same relative abundance and type of genera as the 1 ng sample. The legacy Accel-Amplicon 16S+ITS panel and the new xGen 16S Amplicon Panel v2 products gave similar results when comparing the 2 x 150 and 2 x 300 PE sequencing read percentages; a comparable number of genera were identified from swine manure samples (right table).

Table 1. Reliable results across variety of population groups and sex.

DNA	Sex	Race/population group	% On target	% Coverage uniformity
NA00897	M	Caucasian	99.7	98.2
NA11496	F	Caucasian	96.2	100
NA14624	M	Caucasian	99.9	93.3
NA24143	F	Caucasian (Ashkenazi)	99.8	93.4
NA14639	F	Caucasian (Ashkenazi)	99.9	93.3
NA14628	F	Caucasian (English/German)	99.9	93.3
NA14637	F	Caucasian (French Canadian)	99.9	94.5
NA14634	M	Caucasian (Norwegian)	99.9	95.6
NA12878	F	Caucasian (Utah/Mormon)	99.8	99.3
NA12249	F	Caucasian (Utah/Mormon)	99.9	93.3
HG02190	F	Chinese (Dai)	99.9	93.3
NA24695	F	Chinese (Han)	99.9	92.2
NA19240	F	Yoruban	99.7	97.1

Sequencing metrics for the xGen Sample ID Amplicon Panel with an array of Coriell repository DNA samples (10 ng input each) of different population groups and sex. Libraries were sequenced on a MiniSeq® (Illumina) instrument. On-target and coverage uniformity were calculated as per base.

Chr	POS	SNP ID	REF	ALT	Coriell NA1278	Coriell NA00897	Coriell NA11496
1	67861520	rs2229546	C	A		100%	52%
1	158582646	rs2251969	T	C			100%
1	167849414	rs203849	A	G	50%	100%
1	179520506	rs1410592	G	A	100%	53%	100%
1	209811886	rs2076356	T	G	53%		52%
1	209968684	rs2013162	C	A	100%		47%
1	228431095	rs1771455	A	G	47%	100%	53%
2	44502788	rs3738985	A	C	100%	100%	100%
2	49381585	rs1394205	C	T	47%
2	75115108	rs10194657	A	G			50%
2	169789016	rs497692	T	C		100%
2	170092395	rs2229267	A	G	52%	47%	51%
2	179454394	rs1560221	A	G		47%	48%
2	179455207	rs2163009	T	C		45%	48%
2	215820013	rs10498027	G	A	45%
2	219941063	rs897477	G	A	100%	100%	100%
2	227896976	rs10203363	C	T		100%	46%
3	4403767	rs2819561	A	G	50%	100%	51%
3	4712413	rs2306875	G	A	48%	52%	45%
3	45989044	rs2234358	T	G	49%	100%
3	148727133	rs4938	G	A	52%	51%	46%
4	5749904	rs386594666	T	C		50%	49%
4	86844835	rs6824722	A	G	47%
4	86915848	rs10003909	T	C	51%		50%
4	88534235	rs2736982	A	G	100%		100%
5	13719022	rs30169	T	G	100%	100%	48%
5	13829799	rs1348689	G	A	50%	50%	100%
5	55155402	rs1009639	C	T	99%	99%	99%
5	82834630	rs309557	T	C		53%	54%
5	135392426	rs4669	T	C		48%
5	138456815	rs3088052	T	C	48%	52%	52%
6	146755140	rs2942	G	A	46%	47%	49%
6	152464839	rs2256135	A	G	51%	52%	49%
7	34009946	rs10265207	C	T	100%		99%
7	48450157	rs17548783	T	C	47%	100%
7	50742180	rs1800504	C	T	50%	50%	100%
7	100804140	rs1048303	C	T	52%	100%
7	127250907	rs386607686	T	C	46%	100%	100%
8	94935937	rs4735258	T	C	100%		51%
8	104337096	rs3808554	A	G	48%	47%	45%
9	27202870	rs386602523	A	G	50%		46%
9	77415284	rs7859201	A	C	100%
9	97365642	rs9695	G	A	47%	51%
9	100190780	rs1381532	A	G	100%		100%
9	104184022	rs4577	G	A	100%
9	136304497	rs3124768	A	G	100%	47%	52%
10	69926097	rs2673794	T	C	47%	100%
10	73856984	rs3312	A	G		45%
10	78944590	rs1131824	G	A	47%	49%	100%
10	85972043	rs10749482	A	G	51%	47%	46%
10	95791763	rs17109674	G	A			50%
10	104596924	rs6163	C	A		47%
10	104814162	rs2275271	T	C	46%
10	105819956	rs805701	G	A	100%	100%	100%
10	113920465	rs2277207	G	A	48%	47%	47%
11	6629665	rs1043388	C	T			47%
11	16133413	rs4617548	A	G	100%		45%
11	30255185	rs386601843	C	T	51%	48%	100%
12	993930	rs7300444	C	T
12	8757481	rs2028373	G	A		53%	41%
12	52200742	rs60637	C	A	50%	100%	100%
14	50769717	rs2297995	G	A		100%	100%
14	64637147	rs7161192	C	A		47%
14	74992800	rs699374	A	G	51%		45%
14	76045858	rs2287016	G	A
15	34528948	rs4577050	G	A	100%	47%	46%
15	89401615	rs3825994	T	G	100%	48%	100%
15	89402596	rs698621	T	G	53%	48%	100%
16	68713730	rs2296409	G	A	51%	100%	100%
16	68713823	rs2296408	C	A	49%	100%	100%
16	68729785	rs17715450	C	A	48%	100%	100%
16	70303580	rs2070203	G	A	47%	48%	46%
16	70546234	rs3762171	G	A	48%	45%	50%
17	7192091	rs222842	C	T	47%	100%	100%
17	10536018	rs2285479	G	A	100%		100%
17	10542471	rs2285475	T	G	100%		100%
17	42449789	rs5910	G	A	100%	48%
17	71192663	rs1052706	G	A	100%	51%	55%
17	71192873	rs11544800	A	G	100%	53%	52%
17	71197748	rs1037256	G	A	100%	49%	56%
18	12351342	rs11080572	C	T	47%	100%	52%
18	47455923	rs2298628	C	T	48%	52%	100%
19	10267077	rs2228611	T	C	48%	52%	100%
19	12989560	rs2293682	G	A	47%	51%
19	13445208	rs2248069	C	T	100%	100%	46%
19	16591464	rs9305079	G	A	100%	50%	49%
19	38994910	rs2229144	G	A			51%
20	2413320	rs2076652	T	C	52%	100%	51%
20	6100088	rs10373	A	G	51%	45%	47%
20	19970705	rs2076584	C	T	100%	100%	51%
20	35865054	rs4608	C	T	100%	48%	100%
21	44323590	rs4148973	T	G	48%	51%	48%
21	46908355	rs11702425	T	C	51%	51%	47%
21	47773103	rs2249057	C	A	100%	47%	50%
22	21141300	rs4675	T	C	51%	51%	48%
22	37469591	rs4820268	G	A	51%	100%	50%

The xGen Sample ID Amplicon Panel was used with 10 ng of gDNA from different Coriell samples to create libraries. Sequencing was performed using MiSeq® V2 Reagents. SNP allele frequencies were determined using GATK HaplotypeCaller (Broad Institute). Homozygous SNPs are indicated in blue and heterozygous SNPs are indicated in green. Positions on GHCh37 (hg19). Clear differences are observed between the DNA samples, demonstrating the genotype determination possible with xGen Sample ID Amplicon Panel.

Amplicon distribution per chromosome

Amplicons per chromosome in xGen Sample ID Amplicon Panel consists of 104 amplicons, 95 of which cover known exonic SNPs, and 9 are for sex identification. This coverage gives a discrimination power of 1:85,000 [1].

Chr	#amps
X	1
Y	8
1	8
2	21
3	5
4	5
5	7
6	3
7	6
8	3
9	7
10	9
11	4
12	4
13	1
14	5
15	4
16	6
17	8
18	3
19	5
20	5
21	4
22	3

Figure 1. The ARTIC SARS-CoV-2 Amplicon Panel’s genome coverage for Omicron variants. Two nasopharyngeal swab samples (Cts 28.4 and 13.6) were subjected to the protocol using the ARTIC v4.1 primer pool to create amplicons, then the xGen DNA Library Prep MC Library Prep Kit to adapt the amplicons to create an NGS library. Libraries were sequenced on a MiSeq™ (Illumina) with 2 x 250 bp PE reads. Data was down sampled to 100,000 reads. Alignment was then performed using the Burrows-Wheeler Alignment (BWA) Tool [4] and a consensus file uploaded to Pangolin for lineage calling. High genomic coverage for the BA.1 lineage was observed (IGV screenshot above) indicating the compatibility of the panel with Omicron variants.

Table 2. Sequencing metrics for ARTIC v4.1 libraries created from nasopharyngeal swabs.

Sample	Ct	Reads aligned	On-target	Coverage uniformity	% Genome >10X	Lineage
Sample 1	28.4	100,000	99.7	85.9	97	BA.1
Sample 2	13.6	100,000	99.7	97.6	100	BA.1

Sequencing metrics for NGS libraries originating from nasopharyngeal swabs. Libraries were created with the ARTIC v4.1 primer pool and the xGen DNA Library Prep MC Library Prep Kit. Libraries were sequenced on a MiSeq™ (Illumina) with 2 x 250 bp PE reads. Data was down sampled to 100,000 reads. Lineages were called with Pangolin.

As previously noted, the Ct value for the viral RNA in the sample is a critical metric to obtain usable data. IDT researchers used the SARS-CoV-2 Midnight Amplicon Panel in combination with the IDT xGen DNA Library Prep Kit EZ on samples obtained from nasopharyngeal swabs.

In a proof-of-concept test, sequencing libraries were first prepared according to the SARS-CoV-2 Midnight Amplicon Panel protocol for samples with varying Ct values. These libraries were then subjected to enzymatic fragmentation and library preparation according to the xGen DNA Library Prep Kit EZ recommendations (Figure 3).

Amplicon sequencing combining the xGen SARS-CoV-2 Midnight Amplicon Panel and the xGen Lotus DNA Library Prep provided >99.5% coverage at >5X of the SARS-CoV-2 genome for Ct values <32.

Figure 3. Amplicon sequencing combining the SARS-CoV-2 Midnight Amplicon Panel and the xGen DNA Library Prep Kit EZ provided >99.5% coverage at >10X of the SARS-CoV-2 genome for Ct values 14−19. Data was generated from three independent samples of nasopharyngeal (NP) swab material with three different Ct values: 14.1, 19.9, and 15.3. Libraries were generated following the xGen DNA Library Prep Kit EZ recommendations. The resulting libraries were sequenced with MiSeq™ 300 cycle kit (2 x 150 reads) (Illumina). Samples were subsampled to 197,000 total reads. 100.0% of the reads were aligned to the viral genome and the coverage uniformity ranged from 81−91.9% with a mean coverage of ~950X. Pangolin analysis indicated all samples were Omicron strain (BA.1).

To evaluate the xGen HS EGFR Pathway Amplicon Panel, two DNA standards from Seracare were used. These standards contain mutations with known allele frequencies at either 0.5% or 0.25%. Duplicate SeraSeq® ctDNA Reference Material AF (LGC SeraCare) samples at 10 ng were generated with the 0.5% standard and duplicate SeraSeq® ctDNA Reference Material AF (LGC SeraCare) samples at 20 ng were generated with the 0.25% standard, a total of 4 libraries are shown. As seen in Table 1, the observed allele frequency (AF) aligned with the expected allele frequency for the mutations in the reference cfDNA that were targeted by the xGen HS EGFR Pathway Amplicon Panel. Samples were sequenced on a MiniSeqTM (Illumina).

Table 1: Observed vs. expected allele frequency in NGS libraries prepared using the xGen HS EGFR Pathway Amplicon Panel

				10 ng SeraSeq cfDNA with AF of 0.5%			20 ng SeraSeq cfDNA with AF of 0.25%
Gene	Mutation	Chr	Position	Expected AF	Observed AF (Rep 1)	Observed AF (Rep 2)	Expected AF	Observed AF (Rep 1)	Observed AF (Rep 2)
BRAF	p.V600E	7	140453136	0.5%	0.19%	0.45%	0.25%	0.18%	0.18%
EGFR	p.T790M	7	55249071	0.5%	0.43%	0.57%	0.25%	0.33%	0.36%
EGFR	p.L858R	7	55259515	0.5%	0.86%	0.43%	0.25%	0.26%	0.25%
KRAS	p.G12D	12	25398284	0.5%	0.59%	0.47%	0.25%	0.20%	0.48%
NRAS	p.Q61R	1	115256529	0.5%	0.91%	0.56%	0.25%	0.30%	ND
EGFR	p.E746-A750 (delELREA)	7	55242265	0.5%	0.24%	0.61%	0.25%	0.11%	0.07%
EGFR	p.D770-N771 (ins)	7	55249012	0.5%	0.43%	0.57%	0.25%	0.32%	0.36%

xGen SARS-CoV-2 Amplicon Panels, previously known as Swift Normalase™ Amplicon Panel (SNAP) SARS-CoV-2 enables researchers to identify SARS-CoV-2 strains, including variants, by creating next generation sequencing (NGS) libraries. The sequencing data can then answer research questions, such as:

Which viral strains account for most infections within a local population?
What new strains are emerging?

IDT’s predesigned amplicon panels for COVID research can aid in identifying the extent and pattern of viral spread within a population and allow researchers to quantify the effectiveness of intervention measures.

The xGen SARS-CoV-2 Amplicon Panel enables identification of SARS-CoV-2 variants from a wide array of research sample types such as nasopharyngeal/oropharyngeal swabs, sputa, stool, and wastewater. This panel covers 99.7% of the SARS-CoV-2 genome for identification of mutations from Alpha and Delta strains (see Product Data).

The xGen SARS-CoV-2 Sgene Amplicon Panel provides 100% coverage of the gene which encodes for the spike protein in the virus that causes COVID-19. Identify the presence of emerging viral strains which contain mutations in the Sgene and model potential changes in transmission patterns based upon those mutations.

The xGen ACE2 Gene Amplicon Panel contains 41 amplicons with an average size of 150 bp that provides comprehensive coverage of all coding regions of ACE2. Sequencing ACE2 has the potential to provide insight into disease outcome and facilitate further epidemiological investigations.

cDNA-to-sequencer in 3 hours

To sequence SARS-CoV-2 viral genomic material, first prepare first- or second-strand cDNA from samples such as nasopharyngeal/oropharyngeal swabs, sputa, stool, or wastewater (Figure 1). Generate an NGS library using tiled primer pairs in a single tube to target the 29.9 kb viral genome. Primers were designed against the NCBI Reference Sequence NC_045512.2 (severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1, complete genome) (Table 1). Following a positive result by qPCR, use excess cDNA to perform variant calling and identify the viral strain.

One-tube workflow prepares normalized libraries from cDNA in 3 hours by replacing qPCR library quantification with Normalase technology.

Figure 1. One-tube workflow prepares normalized libraries from cDNA in 3 hours by replacing qPCR library quantification and normalization with the proprietary Normalase™ technology (included in the kit).

Table 1. NGS for SARS-CoV-2 specifications.

Features	Specifications
Design coverage and panel information	99.7% (29,828 of 29,903 total bases) 345 amplicons, sized 116–255 bp (average 150 bp)
Input material	1st or 2nd strand cDNA Minimum 10–100+ viral copies (qRT-PCR Ct value 30–40)
Time	2 hours cDNA-to-Library
Time	3 hours cDNA-to-Normalized-Library Pool
Multiplexing capability	Up to 384 CDIs Up to 1536 UDIs
Compatible with other indexes?	Yes
Recommended depth	Identification: 50K reads per library Variant calling: 1M reads per library

xGen Amplicon Panels for oncology and inherited diseases research utilize multiple overlapping amplicons in a single tube, using a rapid, 2-hour workflow to prepare ready-to-sequence libraries in research studies (Figure 1). The PCR1+PCR2 workflow generates NGS libraries for identifying genetic changes in the genes associated with a variety of tissues and inherited diseases (Table 1). The libraries may be quantified with conventional methods such as Qubit® (Thermo Fisher Scientific) or Agilent Bioanalyzer® and normalized by manual pooling or normalized enzymatically with the included xGen Normalase™ reagents.

Amplicon sequencing libraries made with xGen Custom Amplicon Panels can be created in about 2 hours.

Figure 1. xGen Amplicon Panels have a single tube workflow that is done in as little as 2 hours. Creating an NGS library starts with multiplex PCR. Your custom or predesigned panel is combined with the DNA sample to amplify the targets of interest. The samples are then amplified with indexing primers to create a functional dual indexed library. As an optional step, the xGen Normalase reagent can be used after pooling multiple libraries to ensure each is equally represented in the final sample for the flowcell.

Table 1. List of xGen Oncology & Inherited Diseases Amplicon Panels

Panel	Number of Genes	Genes
xGen 56G Oncology Amplicon Panel v2, 96 rxn	56	ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, DDR2, DNMT3A, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, FOXL2, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, KRAS, MAP2K1, MET, MLH1, MPL, MSH6, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, TSC1, VHL
xGen 57G Pan-Cancer Amplicon Panel, 96 rxn	57	ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, DDR2, DNMT3A, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, FOXL2, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, KRAS, MAP2K1, MET, MLH1, MPL, MSH6, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, TSC1, TSC2, VHL
xGen BRCA1 BRCA2 Amplicon Panel, 96 rxn	2	BRCA1, BRCA2
xGen BRCA1 BRCA2 PALB2 Amplicon Panel, 96 rxn	3	BRCA1, BRCA2, PALB2
xGen CFTR Amplicon Panel, 96 rxn	1	CFTR (All exons including 5’ and 3’ UTRs, select intronic regions (1, 12, 22, and 25))
xGen EGFR Pathway Amplicon Panel, 96 rxn	4	EGFR, BRAF, KRAS, NRAS
xGen Colorectal Amplicon Panel, 96 rxn	16	AKT1, APC, BRAF, ERBB2, ERBB4, KIT, KRAS, NOTCH1, NRAS, PDGFRA, PIK3CA, POLE, PTEN, SMAD4, STK11, TP53
xGen TP53 Amplicon Panel, 96 rxn	1	TP53
xGen Lung Amplicon Panel, 96 rxn	17	AKT1, ALK, ARAF, BRAF, EGFR, ERBB2, ERBB4, FGFR1, FGFR2, FGFR3, KRAS, MAP2K1, MET, NRAS, PIK3CA, PTEN, TP53
xGen Lynch Syndrome Amplicon Panel, 96 rxn	4	MLH1, MSH2, MSH6, PMS2
xGen Myeloid Amplicon Panel, 96 rxn	23	ASXL1, CALR, CEBPA, CSF3R, DNMT3A, EZH2, FLT3, HRAS, IDH1, IDH2, JAK2, JAK3, KDM6A, KIT, MPL, NPM1, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1

Bold indicates whole CDS coverage

IDT is committed to providing quality products to researchers working on the cutting edge of scientific discovery. The xGen Monkeypox Virus Amplicon Panel was designed as part of the next generation sequencing (NGS) Tech Access program at IDT, which is intended to accelerate innovation by enabling earlier access to our most advanced research tools. Tech Access products have not been through our standard, rigorous development cycle. These products are particularly well suited for researchers who require the most up-to-date technology to unlock new discoveries.

The current variants of monkeypox that are circulating have a genome of nearly 200 kb double-stranded DNA [1]. Surveillance of the virus and any potential mutations have gained international support due to the lessons learned through the COVID-19 pandemic. The IDT xGen Monkeypox Virus Amplicon Panel helps to enable researchers to track monkeypox strains, including potential new variants, by next generation sequencing.

DNA-to-sequencer in 2.5 hours

The workflow for the xGen Monkeypox Virus Amplicon Panel starts with extracted viral DNA (Figure 1). You can then generate an NGS library in a single tube using tiled primer pairs designed to target 184 kb of the monkeypox genome. Primers were designed for the currently circulating strain of the monkeypox virus (NCBI accession number ON568298 [1]), which allows you to generate overlapping amplicons in a single-tube, PCR 1 + PCR 2 workflow. If pooling multiple samples for NGS, the xGen Amplicon Core Kit includes the reagents for Normalase™ technology, the proprietary enzymatic normalization step that reduces hands-on time needed for manual normalization. Specifications of the Tech Access, xGen Monkeypox Virus Amplicon Panel are found in Table 1.

Figure 1. xGen Monkeypox Virus Amplicon Panel workflow. A dual-index library is prepared from viral samples in three main steps: 1) multiplex PCR, 2) adapter attachment with indexing PCR, and 3) an optional Normalase™ step to produce equimolar library pools.

Table 1. Features of the xGen Monkeypox Virus Panel.

Features	Specifications
Design coverage and panel information	Comprehensive coverage from positions 6760–190,905. (ITRs not included) 1892 amplicons, sized 93–246 bp (average size is 150 bp)
Input Material	Extracted viral DNA Suggested minimum of 300 viral genome copies
Time	~2.5 hours for viral DNA-to-library
Multiplexing capability	Up to 1536 UDIs
Compatible with other indexes?	Yes
Recommended depth	Strain identification or variant calling: 500K reads per library

The xGen 16S Amplicon Panel v2 and the xGen ITS1 Amplicon Panel offer a reliable NGS workflow that provides coverage and NGS quality data on Illumina® sequencing platforms. These kits leverage multiplex PCR technology, enabling library construction from DNA using tiled primer pairs to target V1-V9 variable rRNA regions and the ITS1 region, each with a single pool of multiplexed primer pairs.

These xGen Amplicon Panel kits utilize multiple overlapping amplicons in a single tube, using a rapid, 2-hour sequencing workflow to prepare ready-to-sequence libraries for research studies (Figure 1). The single pool multiplex PCR workflow generates robust libraries, even from low-input quantities. The libraries may be quantified with conventional methods such as Qubit® (Thermo Fisher Scientific) or Bioanalyzer® (Agilent) and normalized by manual pooling or may be normalized enzymatically with the included xGen Normalase™ reagents. (Note: using the Normalase reagent adds additional time to complete the workflow).

In addition, both the xGen 16S Amplicon Panel v2 and the xGen ITS1 Amplicon Panel facilitate NGS analysis of complex microbial communities (e.g., bacteria, archaea, fungi) using single primer pools that target the 16S rRNA gene (variable regions 1–9) and ITS1 region, respectively. The panels can be customized with additional targets, including antibiotic resistance or virulence genes, allowing sub-genera level identification and functional analysis.

Figure 1. xGen Amplicon Panels have a single tube workflow that completes in 2 hours. Creating an NGS library starts with multiplex PCR. Your panel is combined with the DNA sample to amplify the targets of interest. The samples are then amplified with indexing primers to create a functional dual indexed library. As an optional step, the xGen Normalase reagent can be used after pooling multiple libraries to ensure each is equally represented in the final sample for the flowcell.

The xGen Sample ID Amplicon Panel is a streamlined amplicon NGS prep kit that includes 95 primer pairs targeting exonic single nucleotide polymorphisms (SNPs) with high minor allele frequency (MAF) and 9 amplicons to determine sex. This panel is compatible with circulating, cell-free DNA (cfDNA), as well as formalin-fixed paraffin embedded (FFPE) samples. With the advent of liquid biopsy assays of circulating nucleic acids in oncology research studies, proper tracking of samples has become increasingly critical. This panel represents a powerful and reliable method for tracking and identifying markers in a variety of sample types including liquid biopsy samples.

The power of discrimination of this panel is over 1 in 85,000 [1], making this product suitable for longitudinal studies and scenarios in which genetic fingerprinting is relevant to research design and analysis in various areas including genotyping, research on noninvasive prenatal testing (NIPT), research on transplant rejection, and others. This product is a complete kit that includes all elements necessary to generate multiplex libraries compatible with Illumina® sequencing platforms, including primer pairs and indexed sequencing adapters.

As seen in Figure 1, xGen Sample ID Amplicon Panels have a single tube workflow that is done in as little as 2 hours.

Figure 1. xGen Sample ID Amplicon Panels have a single tube workflow that is done in as little as 2 hours. Creating an NGS library starts with multiplex PCR. Your panel is combined with the DNA sample to amplify the targets of interest. The samples are then amplified with indexing primers to create a functional dual indexed library. As an optional step, the xGen Normalase™ reagent can be used after pooling multiple libraries to ensure each is equally represented in the final sample for the flowcell.

Specifications

Features	xGenTM Sample ID Amplicon Panel
Input DNA required	10–25 ng
Time required	2 hours
Number of amplicons	104
Amplicon size	120–160 (Average 145 bp)
On target percentage	>95%
Coverage uniformity at >20% of mean	>95%
Multiplexing on MiSeq® v2 Nano at 500X depth	38 Samples
Multiplexing on MiSeq® v2 at 750X depth	384 Samples
Sample compatibility	cfDNA and FFPE

Using next generation sequencing (NGS) to analyze SARS-CoV-2 can help researchers understand COVID-19. IDT, in partnership with the ARTIC Network, is offering their latest design of the ARTIC SARS-Co-V-2 Amplicon Panel to support global availability of this important research tool.

The ARTIC network—a group of researchers who are developing a system for processing different virus samples to generate public health information—has developed primer sequences, experimental recommendations, and bioinformatic resources to facilitate NGS analysis of the SARS-CoV-2 genome. Their primer design pipeline (PrimalSeq), previously deployed for other infectious disease sequencing, was especially useful early in the SARS-COV2 pandemic [1].

ARTIC provides a detailed experimental protocol featuring a set of primers for amplicon sequencing to quickly sequence SARS-CoV-2 [2]. They also maintain a resource that alerts researchers to updates in the design [3]. An adaptative protocol that allows for sequencing on Illumina sequencers have been developed by others [4].

The sequence of the SARS-CoV-2 genome can enable research in many areas, including:

Origin identification
Mutations and viral evolution rates
Transmission routes of variants
Population surveillance, such as wastewater-based epidemiology.

DR. JOSHUA QUICK
University of Birmingham
ARTICnetwork

The ARTIC network is delighted to be partnering with IDT to produce primer pools for SARS CoV 2 genome sequencing. The ARTIC nCoV 2019 amplicon sequencing protocol has been widely adopted across the world, and the genome data is critical to understanding and tracking the outbreak. Our mission is to put genomics at the heart of outbreak response and having this resource available will help more groups establish genome sequencing capabilities in their own labs in a cost-effective and reproducible way.

Workflow

The ARTIC SARS-CoV-2 Amplicon Panel workflow includes cDNA synthesis, amplicon generation, and cleanup. The final amplicons average 400 bp require 2 x 250 bp read lengths on either the NovaSeq™ or MiSeq™ sequencers (Illumina).

Table 1. Specifications for the ARTIC SARS-CoV-2 Amplicon Panel

Recommended Ct value	Coverage	Contiguous amplicons	Coordinates covered
<30	99.7%	99	50-29,827

The Midnight Panel was created by Drs. Nikki Freed and Olin Silander of Massey University in New Zealand [1] and recently updated by Dr. John Tyson and Tracy Lee of the British Columbia Centre for Disease Control. This panel consists of 29 amplicons of approximately 1200 bp in length (Figure 1). The reduced number of amplicons allows for more consistent sequencing across the genome than would be achievable with a higher number of amplicons.

xGen SARS-CoV-2 Midnight Amplicon Panel amplicons.

Figure 1. SARS-CoV-2 Midnight Amplicon Panel amplicons. The multiplex primer panel consists of two pools. The spacing is designed to tile across the entire SARS-CoV-2 genome, generating 29 amplicons approximately 1200 bp in length.

Since the primers only anneal to a total of 4.5% of the viral genome, the chance that viral mutations will disrupt primer annealing is lower. This results in a lowered chance of amplicon dropouts.

The Ct value for the amplicon sequencing reaction is an important metric since the higher the Ct, the lower the number of viral genomes in the sample. Samples with less viral genomic material pose difficulty for primer annealing and amplification.

In our experiment, contrived samples were prepared by mixing inactivated SARS-CoV-2 viral material into a negative background matrix of a negative nasopharyngeal (NP) swab in viral transport medium (VTM), with two samples per Ct value. Results show that when the SARS-CoV-2 Midnight Amplicon Panel and xGen DNA Library Prep Kit EZ are combined, the recommended Ct value is <32 which="" has="" been="" shown="" to="" sequence="" 97="" of="" the="" sars-cov-2="" genome="" with="" sufficient="" depth="" identify="" variants="" table="" 1="" p="">

Table 1. SARS-CoV-2 Midnight Amplicon Panel and xGen DNA Library Prep Kit EZ metrics.

Recommended Ct value	Coverage (%)	Contiguous amplicons (count)	Coordinates covered
<32	97%	29	30–28,985

Workflow

Since the length of the amplicons created by the SARS-CoV-2 Midnight 1200 Amplicon Panel are 1200 bp, Illumina® sequencing platforms are not compatible with this workflow. To use Illumina sequencing platforms, the SARS-CoV-2 amplicons generated with the Midnight panel can be fragmented using the xGen DNA Library Prep Kit EZ (with enzymatic DNA fragmentation). An overview of this workflow shows the steps necessary for library construction (Figure 2). IDT offers many of the reagents for the workflow, from the SARS-CoV-2 Midnight Panel and the components for library construction, to Nuclease-Free Water.

Figure 2. Expected experimental workflow for SARS-CoV-2 genome sequencing using the SARS-CoV-2 Midnight Amplicon Panel in combination with the xGen DNA Library Prep Kit EZ. By using the xGen DNA Library Prep Kit after the SARS-CoV-2 Midnight Amplicon Panel, the original 1200 bp fragments are converted into a library that is compatible with Illumina sequencers and chemistry.

For labs with access to Oxford Nanopore Sequencing instruments, the amplicons created using the SARS-CoV-2 Midnight Amplicon Panel can be prepared using one of the barcoding kits available directly from Oxford Nanopore (e.g., Rapid Barcoding Kit, SQK-RBK004).

The IDT xGen HS EGFR Pathway Amplicon Panel covers contiguous regions of the EGFR gene, and hotspot coverage of BRAF, KRAS, and NRAS with a panel of amplicon-generating primers. The final amplicons average only 136 bp making them compatible with DNA fragments from cell-free DNA (cfDNA) and/or FFPE DNA. The final target size of this panel is 1.5 kbp.

As seen in Figure 1, this workflow includes three PCR steps—one for the incorporation of unique molecular identifiers (UMIs) (PCR I), one for target amplification (PCR II), and one for the addition of combinatorial dual indexed adapters (PCR III), enabling multiplexing of up to 96 unique libraries. Bead-based SPRI® cleanups (Beckman Coulter) are used to purify the sample by removing unused oligonucleotides and changing buffer composition between steps.

Figure 1. The xGen HS EGFR Pathway Amplicon Panels are used to prepare indexed Illumina®-compatible libraries from cfDNA or FFPE DNA. The workflow for preparing the final targeting sequencing libraries includes PCR I, PCR II, and PCR III. The first amplification uses the panel of primers to amplify the targeted genes from the EGFR pathway. The primers also incorporate UMIs. A second amplification step increases the total number of fragments for indexing, while the third PCR completes the indexing.

PRODUCT DATA

Table 1: Observed vs. expected allele frequency in NGS libraries prepared using the xGen HS EGFR Pathway Amplicon Panel

				10 ng SeraSeq cfDNA with AF of 0.5%			20 ng SeraSeq cfDNA with AF of 0.25%
Gene	Mutation	Chr	Position	Expected AF	Observed AF (Rep 1)	Observed AF (Rep 2)	Expected AF	Observed AF (Rep 1)	Observed AF (Rep 2)
BRAF	p.V600E	7	140453136	0.5%	0.19%	0.45%	0.25%	0.18%	0.18%
EGFR	p.T790M	7	55249071	0.5%	0.43%	0.57%	0.25%	0.33%	0.36%
EGFR	p.L858R	7	55259515	0.5%	0.86%	0.43%	0.25%	0.26%	0.25%
KRAS	p.G12D	12	25398284	0.5%	0.59%	0.47%	0.25%	0.20%	0.48%
NRAS	p.Q61R	1	115256529	0.5%	0.91%	0.56%	0.25%	0.30%	ND
EGFR	p.E746-A750 (delELREA)	7	55242265	0.5%	0.24%	0.61%	0.25%	0.11%	0.07%
EGFR	p.D770-N771 (ins)	7	55249012	0.5%	0.43%	0.57%	0.25%	0.32%	0.36%

Chr	#amps
X	1
Y	8
1	8
2	21
3	5
4	5
5	7
6	3
7	6
8	3
9	7
10	9
11	4
12	4
13	1
14	5
15	4
16	6
17	8
18	3
19	5
20	5
21	4
22	3

Chr	#amps
X	1
Y	8
1	8
2	21
3	5
4	5
5	7
6	3
7	6
8	3
9	7
10	9
11	4
12	4
13	1
14	5
15	4
16	6
17	8
18	3
19	5
20	5
21	4
22	3

WORKFLOW-K

xGen™ Oncology & Inherited Diseases Amplicon Panels

xGen™ Metagenomics Amplicon Panels

xGen™ SARS-CoV-2 Amplicon Panels

xGen™ Monkeypox Virus Amplicon Panel

xGen™ Sample Identification (ID) Amplicon Panel

ARTIC SARS-CoV-2 Amplicon Panel

xGen™ SARS-CoV-2 Midnight Amplicon Panel

xGen™ HS EGFR Pathway Amplicon Panel

Full genomic coverage of lineages with a single primer set

Research Investigation

Case Study I Genemarkers, LLC

Obtain genomes from viral titers as low as 10–100 viral copies

Coverage and on-target mapping rates

Sequencing results from titers as low as 300 viral genome copies

Super amplicons help maintain sequencing coverage despite mutations

Coverage of all variable regions of the 16S rRNA gene and ITS1 region

xGen 16S Amplicon Panel v2 (primers)

xGen ITS1 Amplicon Panel (primers)

The xGen 16S Amplicon Panel v2 and the xGen ITS1 Amplicon Panel provide representation of diverse microbial communities

Consistent function with varying biomass, sample types, and read length

Amplicon distribution per chromosome

cDNA-to-sequencer in 3 hours

DNA-to-sequencer in 2.5 hours

Specifications

Workflow

Workflow

PRODUCT DATA

Targeted amplicon library prep kit for contiguous coverage (overlapping amplicons), comprises multiplexed PCR, indexing PCR, and Normalase™ reagents (target-specific panel and indexing primers sold separately).

xGen™ COVID Amplicon Panels

COVID panels that utilize multiple overlapping amplicons in a single tube, comprises a premixed target-specific primer pool.

xGen™ Amplicon UDI Primers

Premixed Normalase unique dual index primer pairs for sample indexing during library amplification and conditioning for Normalase enzymology, 1536 ten base index pairs dispensed into 16 96-well plates, Reagent R7 NOT provided, instead uses reagent supplied in the xGen Amplicon Core Kit.

xGen™ PEG NaCl Buffer

Buffer used during ''with bead'' purification steps when DNA is eluted into the next reaction mixture and the beads are reused. Supplied with the xGen Amplicon Core Kit but also sold separately if additional buffer is needed.

Gen™ Amplicon CDI Primers

Individual combinatorial dual index primers for sample indexing during library amplification, 20 eight-base index primers dispensed separately for 96-plex combinations.

xGen™ Oncology/Inherited Disease Amplicon Panels

Oncology and infectious disease panels that utilize multiple overlapping amplicons in a single tube; comprises a premixed target-specific primer pool.

xGen™ Monkeypox Virus Amplicon Panel

Monkeypox Virus panel that utilizes multiple overlapping amplicons in a single tube, comprises a premixed target-specific primer pool.

xGen™ Metagenomics Amplicon Panels

Metagenomics panels that utilize multiple overlapping amplicons in a single tube; comprises a premixed target-specific primer pool.

xGen™ Sample ID Amplicon Panel

Sample ID panel that utilizes multiple overlapping amplicons in a single tube; comprises a premixed target-specific primer pool.

ARTIC nCoV-2019 Amplicon Panel

Predesigned panel designed in collaboration with the ARTIC network targeting the SARS-CoV-2 genome.

SARS-CoV-2-Midnight-1200 Amplicon Panel

The updated Midnight Panel V2 was designed by Dr. John Tyson and Tracy Lee (BC Centre for Disease Control) together with Dr. Nikki Freed (Massey University and University of Auckland) and Dr. Olin Silander (Massey University)

xGen™ HS Amplicon Panel

EGFR amplicon panel utilizing UMIs for identification of mutations occurring at 0.25% allele frequency. Custom panels available upon request.

Forgalmazott termékeink gyártói - keressen gyártó szerint a logóra kattintva

Chr	#amps
X	1
Y	8
1	8
2	21
3	5
4	5
5	7
6	3
7	6
8	3
9	7
10	9
11	4
12	4
13	1
14	5
15	4
16	6
17	8
18	3
19	5
20	5
21	4
22	3