Yes, the DMD Research Panel v1.0 supports mixed usage with the NEXome-series Panels. The CDS (coding sequence) probes of the DMD Research Panel v1.0 can be separated to avoid overlap with the DMD gene exon probes in the whole-exome panel, thereby eliminating data redundancy.

Fusion genes serve as critical molecular markers for the diagnosis and treatment of hematologic malignancies, making their accurate detection essential. The DNA workflow detects gene fusions by designing probes covering intronic regions; however, due to the large size and high repetitiveness of introns, relying solely on DNA detection poses challenges such as high cost and incomplete coverage, and it cannot confirm the functional impact of fusion events. In contrast, the RNA workflow directly focuses on the transcript level, enabling precise identification of actual fusion events and their partner genes while assessing whether these fusions lead to aberrant transcription. Consequently, the combined use of DNA and RNA workflows provides a “dual safeguard” for fusion gene detection, enhancing both the comprehensiveness and accuracy of the results.

Total RNA-seq requires rRNA depletion and individual reactions per sample, increasing both complexity and cost. Moreover, total RNA-seq exhibits relatively lower sensitivity, particularly in detecting low-frequency fusion events in low-expression samples. In contrast, the DNA panel in NanoHema Panel v2.0 already covers the intronic regions of key genes, allowing users to flexibly add custom intronic regions based on requirements and budget without the need for a separate RNA panel.

NanoHema Panel v2.0 is a comprehensive large panel that covers a wide range of hematologic malignancy-associated gene variants. To meet diverse clinical needs, the panel offers multiple customization options:

•  It can be flexibly split into multiple sub-panels (e.g., for acute myeloid leukemia [AML], lymphoblastic leukemia, T-cell lymphoma, and B-cell lymphoma).

•  Additional target genes can be incorporated based on specific research or diagnostic requirements, enabling the creation of a personalized detection solution.

• Support. Different library preparation approaches can be employed for RNA and DNA viruses. One approach involves the concurrent library preparation of RNA and DNA viruses, followed by subsequent hybridization capture steps. Another approach entails preparing RNA libraries and DNA libraries separately, and then mixing them for hybridization capture. Generally, for RNA libraries, there is no need to perform the step of removing of host ribosomal RNA.
• As shown in Figure, the example demonstrates the capture scenario when RNA libraries containing influenza virus (H10N3) and DNA libraries are hybridized together. A total of 15 libraries are involved, including 11 DNA libraries and 4 RNA libraries. The example library data amount was 100 Mb (totaling 9.3 Gb for all 15 libraries), with host sequences accounting for 58.3%.

Due to significant variations in pathogen content among different samples, if the aim is to achieve similar data amounts for each sample, it’s not feasible to perform multiple-plex hybridization; separate hybridization must be carried out instead. However, as shown in Figure 5, when there are orders of magnitude differences in microbial content among samples, although there are substantial variations in data amounts, low-abundance samples require relatively less data on their own. Moreover, different samples, using the same experimental parameters, exhibit similar PCR duplication rates. If they are subjected to separate hybrid capture and provided with the same sequencing data, low-abundance samples would need an increased number of PCR cycles. The identical information content would only be repeated to form an augmented sequencing data, as post-panel capture sequencing becomes easier to saturate. Based on these considerations, for samples with similar origins, performing the multiple-plex hybridization process is recommended. 

• As shown in Figure 4, the detection limit for samples with varying microbial content is approximately 3 copies. In practical applications, sensitivity is mainly determined by the amount of input for library preparation. It's important to note that the library preparation process is constrained by conversion efficiency, meaning not all copies can be successfully converted.  Similarly, when the copy number is in the single digits, the success rate of multiplex amplicons'  amplification decreases.
• Furthermore, having less than 1 copy does not imply that it cannot be detected. This is due to the fact that when NEX-t Panel v1.0 features multiple probe capture regions for a single species, their copy numbers can accumulate, enhancing the detection sensitivity.

• The on-target rate of pathogens are significantly influenced by the amount of pathogens within the samples. As shown in Figure, under the same host background, as the microbial reference material is gradually diluted, the amount of data decreases proportionally, along with a decrease in on-target rate.
• Although multiple capture rounds can substantially improve on-target rate, this strategy comes with the risk of information loss (dropout). Especially when the amount of pathogen is low, the on-target rate is low and is more l sensitive to dropout. Therefore, careful consideration of the pros and cons is needed when contemplating multiple capture rounds. Additionally, from another perspective, when the pathogen genome copy numbers in the sample are in the single digits, increasing on-target rate merely involves repetitive sequencing of these few copies, yielding limited additional useful information.

Note: Simulated microbial community samples of 0.001% - 1% MSA-1003 were created by diluting a mixture of 20 strains of genomic material (ATCC, MSA-1003) using the human genomic DNA standard (Promega, G1471) at various ratios. Library preparation was performed using 50 ng input with the NadPrep EZ DNA Library Preparation Kit v2, followed by hybrid capture (2-hour hybridization) using NEX-t Panel v1.0 with NadPrep ES Hybrid Capture Reagents. Sequencing platform: Illumina Novaseq6000, PE150. The BWA was used for alignment of raw reads to the reference genome composed of the hg19 human genome and 20 microbial genome reference sequences, and reads distribution was analyzed.

NEX-t Panel v1.0 is recommended for use  in conjunction with NadPrep ES Hybrid Capture Reagents, which significantly shorten the hybridization time while simplifying the experimental steps.Hybrid capture sequencing is more robust and has a lower failure rate than multiplex amplicon sequencing.

• In general, achieving complete probe coverage of pathogen microbiome genomes is impractical. On one hand, designing probes for a single bacterial species can result in hundreds of thousands of probes when considering sequence polymorphism beyond reference genomes.  On the other hand, to prevent off-target effects when there is similarity between pathogenic and host sequences, certain regions must be excluded. Therefore, NEX-t Panel v1.0 emphasizes designing the fewest probes to enable the broadest range of pathogen analysis, resulting in a significantly streamlined panel size compared to tNGS schemes with millions of probes in hybrid capture.
• The number of probes can be roughly equivalent to the number of amplicons in multiplex PCR. However, due to the higher tolerance of probes and the ability to capture sequences flanking the probe, a certain number of probes can provide more diverse information compared to a similar number of multiplex amplicons.
• Compared to multiplex PCR, expanding a hybrid capture panel is simpler as it only requires adding probes. Therefore, NEX-t Panel v1.0 can be conveniently customized and upgraded through combination.