MEGA SAMPLES VOL-96

We rapidly developed a qPCR-based detection method on the basis of the sequence of the receptor-binding domain of the S gene, which was the most variable region of the genome (Fig. 1c). Our data show that the primers could differentiate 2019-nCoV from all other human coronaviruses including bat SARSr-CoV WIV1, which shares 95% identity with SARS-CoV (Extended Data Fig. 4a, b). Of the samples obtained from the seven patients, we found that six BALF and five oral swab samples were positive for 2019-nCoV during the first sampling, as assessed by qPCR and conventional PCR. However, we could no longer detect virus-positive samples in oral swabs, anal swabs and blood samples taken from these patients during the second sampling (Fig. 2a). However, we recommend that other qPCR targets, including the RdRp or envelope (E) genes are used for the routine detection of 2019-nCoV. On the basis of these findings, we propose that the disease could be transmitted by airborne transmission, although we cannot rule out other possible routes of transmission, as further investigation, including more patients, is required.

Routine sequence management and analysis was carried out using DNAStar. The sequence alignment of complete genome sequences was performed using MAFFT (v.7.307) with default parameters. The codon alignments of full-length S and RdRp gene sequences were converted from the corresponding protein alignments by PAL2NAL (v.14); the protein alignments were created by Clustal Omega (v.1.2.4) using default parameters. Maximum likelihood phylogenetic trees were generated using RAxML (v.0.9.0) with GTR+G substitution model and 1,000 bootstrap replicates.

MEGA SAMPLES VOL-96

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Z.-L.S., P.Z., Y.-Y.W. and G.-F.X. conceived the study. X.-G.W., C.-L.H., H.-D.C., F.D., Q.-J.C., F.-X.Z. and L.-L.L. collected patient samples. X.-L.Y., B.Y., W.Z., B.L., J.C., X.-S.Z., Y.L., H.G., R.-D.J., M.-Q.L., Y.C., X.W., X.-R.S. and K.Z. performed qPCR, serology and virus culturing experiments. L.Z., Y.Z., H.-R.S. and B.H. performed genome sequencing and annotations.

a, Standard curve for qPCR primers. The PCR product of the S gene that was serial diluted in the range of 108 to 101 (lines from left to right) was used as a template. Primer sequences and experimental conditions are described in the Methods. b, Specificity of the qPCR primers. Nucleotide samples from the indicated pathogens were used.

a, b, Vero E6 cells are shown at 24 h after infection with mock virus (a) or 2019-nCoV (b). c, d, Mock-virus-infected (c) or 2019-nCoV-infected (d) samples were stained with rabbit serum raised against recombinant SARSr-CoV Rp3 N protein (red) and DAPI (blue). The experiment was conducted twice independently with similar results. e, The ratio of the number of reads related to 2019-nCoV among the total number of virus-related reads in metagenomics analysis of supernatants from Vero E6 cell cultures. f, Virus growth in Vero E6 cells. g, Viral particles in the ultrathin sections were imaged using electron microscopy at 200 kV. The sample was from virus-infected Vero E6 cells. The inset shows the viral particles in an intra-cytosolic vacuole.

TruSeq library prep uses adapter-embedded indexes to enable high throughput processing and application flexibility. The universal, methylated adapter design incorporates an index sequence at the initial ligation step. The same TruSeq kit can be used to prepare samples for single-read, paired-end, and multiplexed sequencing on all Illumina sequencing instruments. Both RNA and DNA preparation kits include adapters containing unique index sequences that are ligated to sample fragments at the beginning of the library construction process. This allows samples to be pooled and then individually identified during downstream analysis. These indexes can generally work with other library preps.

In signal processing, sampling is the reduction of a continuous-time signal to a discrete-time signal. A common example is the conversion of a sound wave to a sequence of "samples".A sample is a value of the signal at a point in time and/or space; this definition differs from the usage in statistics, which refers to a set of such values.[A]

A sampler is a subsystem or operation that extracts samples from a continuous signal. A theoretical ideal sampler produces samples equivalent to the instantaneous value of the continuous signal at the desired points.

The sampling frequency or sampling rate, fs, is the average number of samples obtained in one second, thus fs = 1/T, with the unit samples per second, sometimes referred to as hertz, for example e.g. 48 kHz is 48,000 samples per second.

Most sampled signals are not simply stored and reconstructed. The fidelity of a theoretical reconstruction is a common measure of the effectiveness of sampling. That fidelity is reduced when s(t) contains frequency components whose cycle length (period) is less than 2 sample intervals (see Aliasing). The corresponding frequency limit, in cycles/sec (hertz), is 0.5 cycle/sample fs samples/sec = fs/2, known as the Nyquist frequency of the sampler. Therefore, s(t) is usually the output of a low-pass filter, functionally known as an anti-aliasing filter. Without an anti-aliasing filter, frequencies higher than the Nyquist frequency will influence the samples in a way that is misinterpreted by the interpolation process.[3]

Audio is typically recorded at 8-, 16-, and 24-bit depth, which yield a theoretical maximum signal-to-quantization-noise ratio (SQNR) for a pure sine wave of, approximately, 49.93 dB, 98.09 dB and 122.17 dB.[21] CD quality audio uses 16-bit samples. Thermal noise limits the true number of bits that can be used in quantization. Few analog systems have signal to noise ratios (SNR) exceeding 120 dB. However, digital signal processing operations can have very high dynamic range, consequently it is common to perform mixing and mastering operations at 32-bit precision and then convert to 16- or 24-bit for distribution.

Video digital-to-analog converters operate in the megahertz range (from 3 MHz for low quality composite video scalers in early games consoles, to 250 MHz or more for the highest-resolution VGA output).

The process of volume rendering samples a 3D grid of voxels to produce 3D renderings of sliced (tomographic) data. The 3D grid is assumed to represent a continuous region of 3D space. Volume rendering is common in medical imaging, X-ray computed tomography (CT/CAT), magnetic resonance imaging (MRI), positron emission tomography (PET) are some examples. It is also used for seismic tomography and other applications.

When a bandpass signal is sampled slower than its Nyquist rate, the samples are indistinguishable from samples of a low-frequency alias of the high-frequency signal. That is often done purposefully in such a way that the lowest-frequency alias satisfies the Nyquist criterion, because the bandpass signal is still uniquely represented and recoverable. Such undersampling is also known as bandpass sampling, harmonic sampling, IF sampling, and direct IF to digital conversion.[22]

The most immediate step will be to expand our efforts to recruit editors, editorial board members, and reviewers from diverse backgrounds. In addition, our team has been paying close attention to concerns raised about biases in the evaluation of work that includes samples from under-represented groups or from authors from under-represented backgrounds. For instance, studies with samples from under-represented groups have sometimes been criticized for a lack of generalizability, whereas samples of college students get a pass on this issue (Atherton, 2021). We pledge to watch for these problematic comments in reviews and decision letters to reduce the negative impact that such biases have. Anyone who has concerns about their experiences during the review process can contact the editor-in-chief at any time.

Library normalization is the process of diluting libraries of variable concentration to the same concentration before volumetric pooling, ensuring an even read distribution for all samples. Normalization best practices can be used for any Illumina library preparation requiring a manual normalization. Steps for normalization are:

Validate the library size by running the samples on a Bioanalyzer or Fragment Analyzer. This step also provides visibility into possible library issues, such as adapter dimers or unexpected library sizes.

For the most even representation of samples and most reliable cluster density, make sure that all pipetted volumes are at least 2 µl. Pipetting less than 2 µl can introduce significant concentration errors. Adjust calculations to make sure that appropriate volumes are used. For highly concentrated libraries, use one or more intermediate concentrations.

In December 2019, a cluster of patients with pneumonia of unknown cause was linked to a seafood wholesale market in Wuhan, China. A previously unknown betacoronavirus was discovered through the use of unbiased sequencing in samples from patients with pneumonia. Human airway epithelial cells were used to isolate a novel coronavirus, named 2019-nCoV, which formed a clade within the subgenus sarbecovirus, Orthocoronavirinae subfamily. Different from both MERS-CoV and SARS-CoV, 2019-nCoV is the seventh member of the family of coronaviruses that infect humans. Enhanced surveillance and further investigation are ongoing. (Funded by the National Key Research and Development Program of China and the National Major Project for Control and Prevention of Infectious Disease in China.). 2ff7e9595c