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.

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.">

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.

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.">

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.

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]).">

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]).

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.

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.

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.

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.

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.

 

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).

 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:

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
	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.

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

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.

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

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.

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

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™ Amplicon Core Kit

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.

The xGen SARS-CoV-2 Amplicon Panels enable identification of SARS-CoV-2 variants from research samples such as nasopharyngeal/oropharyngeal swabs, sputa, stool, and wastewater. The xGen ACE2 Gene Amplicon Panel amplifies the angiotensin converting enzyme 2 gene which has been proven to be the SARS-CoV-2 spike protein receptor [1]. The xGen SARS-CoV-2 Sgene panel amplifies the viral gene for the spike protein only.

The xGen SARS-CoV-2 Amplicon Panels support:

• 99.7% genomic coverage†
• Full genomic coverage of lineages from a single primer set
• Sequence data from viral titers as low as 10–100 viral copies
• cDNA-to-sequencer in 3 hours
• Up to 1536 UDIs

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.

• Compatible with Illumina® sequencing platforms
• Designed for research on germline and somatic variant identification, or targeted sequencing
• Offers overlapping amplicons in a fast, easy, single-tube workflow

xGen™ Monkeypox Virus Amplicon Panel

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

Monkeypox has been termed a global health emergency. For researchers interested in tracking the evolution of the monkeypox virus genome, it can be difficult to get sufficient coverage. The xGen Monkeypox Virus Amplicon Panel provides a single-tube, two-step PCR amplification workflow with primer sets designed to create amplicons across the entire genome.

The xGen Monkeypox Virus Amplicon Panel supports:

• Comprehensive coverage from positions 6760–190,905 (ITRs not included)
• Sequence data from viral titers as low as 300 viral genome copies
• Single-tube workflow
• Viral DNA-to-sequencer in ~2.5 hours
• Up to 1536 UDIs
• Super amplicon technology

xGen™ Metagenomics Amplicon Panels

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

• The xGen 16S rRNA v2 Amplicon Panel targets V1–V9 variable regions of the 16S rRNA gene
• The xGen ITS1 Amplicon Panel targets the internal transcribed spacer regions of the rRNA genes
• Integrated library normalization enables streamlined library balancing and pooling without the need to quantify samples
• Unique amplicon chemistry generates diverse clusters without PhiX or phased primers, leading to more efficient sequencing (Figure 4)
• Compatible with Illumina® sequencers and read lengths
• Simple, single tube, 2-hour workflow

xGen™ Sample ID Amplicon Panel

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

• Easily track samples within and between studies
• Ideal for confirming research on tumor/normal pairs
• Power of discrimination over 1 in 85,000 [1]
• Reliable calling of germline variants
• Complements both WGS and exome sequencing for sample tracking
• On-target coverage uniformity >95%
• Leverages the high fidelity of Illumina® platforms
• Complete library generation in a single kit
• Reduce sequencing cost with 1536 index pairs

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)

• Amplifies the SARS-CoV-2 genome for sequencing using premixed multiplex PCR primers
• Provides data for understanding SARS-CoV-2 viral evolution and the epidemiology of COVID-19
• Data identifies SARS-CoV-2 variants, including alpha, beta, gamma, delta, mu, and omicron
• Panel generates 29 tiled amplicons, approximately 1200 bp in length
• Panel is optimized for high throughput applications or automated workflows
• Samples with Ct values <32 are="" recommended="" for="" complete="" genome="" sequencing="" coverage="" based="" on="" initial="" testing="" li="">

xGen™ HS Amplicon Panel

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

• Identifies SNVs and indels down to 0.25% allele frequency
• Compatible with cfDNA and FFPE DNA
• Amplifies from 10–50 ng of cfDNA
• Makes Illumina®-compatible libraries within 3 hours
• Supports research in oncology, graft vs. host disease, and single nucleotide variants in the EGFR pathway

xGen Amplicon Panels for Oncology Research, Inherited Disease Research, and Sample Tracking protokoll

xGen SARS-CoV-2 Amplicon Panel and xGen SARS-CoV-2 S Gene Amplicon Panel protokoll

xGen Amplicon Panels for viral genome sequencing Protokoll

xGen 16S Amplicon Panel v2 and xGen ITS1 Amplicon Panel Protokoll

xGen DNA Library Prep: Sequencing SARS-CoV-2 with the ARTIC nCoV-2019 Amplicon Panel Demonstrated Protokoll

Sequencing SARS-CoV-2 with the Midnight Amplicon Panel Demonstrated Protocol

xGen HS EGFR Pathway Amplicon Panel Protokoll