You asked how is it placed in the gene sequencing machine.
The first investigation on SARS-CoV-2 used bronchoalveolar lavage fluid from a patient as described in this peer reviewed scientific paper.
I’m not sure a layman would find this easy to understand ….
“To investigate the possible aetiological agents associated with this disease, we collected bronchoalveolar lavage fluid (BALF) and performed deep meta-transcriptomic sequencing. The clinical specimen was handled in a biosafety level 3 laboratory at Shanghai Public Health Clinical Center. Total RNA was extracted from 200 μl of BALF and a meta-transcriptomic library was constructed for pair-end (150-bp reads) sequencing using an Illumina MiniSeq as previously described4,6,7,8. In total, we generated 56,565,928 sequence reads that were de novo-assembled and screened for potential aetiological agents. Of the 384,096 contigs assembled by Megahit9, the longest (30,474 nucleotides (nt)) had a high abundance and was closely related to a bat SARS-like coronavirus (CoV) isolate—bat SL-CoVZC45 (GenBank accession number MG772933)—that had previously been sampled in China, with a nucleotide identity of 89.1% (Supplementary Tables 1, 2). The genome sequence of this virus, as well as its termini, were determined and confirmed by reverse-transcription PCR (RT–PCR)10 and 5′/3′ rapid amplification of cDNA ends (RACE), respectively. This virus strain was designated as WH-Human 1 coronavirus (WHCV) (and has also been referred to as ‘2019-nCoV’) and its whole genome sequence (29,903 nt) has been assigned GenBank accession number MN908947. Remapping the RNA-sequencing data to the complete genome of WHCV resulted in an assembly of 123,613 reads, providing 99.99% genome coverage at a mean depth of 6.04× (range, 0.01–78.84×) (Extended Data Fig. 3). The viral load in the BALF sample was estimated by qPCR to be 3.95 × 108 copies per ml (Extended Data Fig. 4).
The viral genome organization of WHCV was determined by sequence alignment to two representative members of the genus Betacoronavirus: a coronavirus associated with humans (SARS-CoV Tor2, GenBank accession number AY274119) and a coronavirus associated with bats (bat SL-CoVZC45, GenBank accession number MG772933). The un-translational regions and open-reading frame (ORF) of WHCV were mapped on the basis of this sequence alignment and ORF prediction. The WHCV viral genome was similar to these two coronaviruses (Fig. 1 and Supplementary Table 3). The order of genes (5′ to 3′) was as follows: replicase ORF1ab, spike (S), envelope (E), membrane (M) and nucleocapsid (N). WHCV has 5′ and 3′ terminal sequences that are typical of betacoronaviruses, with 265 nt at the 5′ terminal end and 229 nt at the 3′ terminal end. The predicted replicase ORF1ab gene of WHCV is 21,291 nt in length and contained 16 predicted non-structural proteins (Supplementary Table 4), followed by (at least) 13 downstream ORFs. Additionally, WHCV shares a highly conserved domain (LLRKNGNKG: amino acids 122–130) in nsp1 with SARS-CoV. The predicted S, ORF3a, E, M and N genes of WHCV are 3,822, 828, 228, 669 and 1,260 nt in length, respectively. In addition to these ORF regions, which are shared by all members of the subgenus Sarbecovirus, WHCV is similar to SARS-CoV in that it carries a predicted ORF8 gene (with a length of 366 nt) that is located between the M and N ORF genes. The functions of WHCV ORFs were predicted on the basis of those of known coronaviruses and are described in Supplementary Table 5. In a manner similar to SARS-CoV Tor2, a leader transcription regulatory sequence (TRS) and nine putative body TRSs could be readily identified upstream of the 5′ end of the ORF in WHCV, and the putative conserved TRS core sequence appeared in two forms—ACGAAC or CUAAAC (Supplementary Table 6).
To determine the evolutionary relationships between WHCV and previously identified coronaviruses, we estimated phylogenetic trees on the basis of the nucleotide sequences of the whole-genome sequence, the non-structural protein genes ORF1a and ORF1b, and the main structural proteins encoded by the S, E, M and N genes (Fig. 2 and Extended Data Fig. 5). In all phylogenies, WHCV clustered with members of the subgenus Sarbecovirus, including the SARS-CoV that was responsible for the global SARS pandemic1,2 of 2002–2003, as well as a number of SARS-like coronaviruses that have been obtained from bats5,11,12,13. However, WHCV changed topological position within the subgenus Sarbecovirus depending on which gene was used, which suggests that recombination has occurred in this group of viruses in the past (Fig. 2 and Extended Data Fig. 5). Specifically, in the S gene tree (Extended Data Fig. 5), WHCV was most closely related to the bat coronavirus SL-CoVZC45 with 82.3% amino acid identity (and around 77.2% amino acid identity to SARS-CoV; Supplementary Table 3) whereas in the ORF1b phylogeny, WHCV fell in a basal position within the subgenus Sarbecovirus (Fig. 2). This topological division, which probably reflects recombination among the bat sarbecoviruses, was also observed in the phylogenetic trees estimated for conserved domains in the replicase polyprotein pp1ab (Extended Data Fig. 6).
To better understand the potential of WHCV to infect humans, the receptor-binding domain (RBD) of its spike protein was compared with those of SARS-CoVs and bat SARS-like CoVs. The RBD sequences of WHCV were more closely related to those of SARS-CoVs (73.8–74.9% amino acid identity) and SARS-like CoVs, including strains Rs4874, Rs7327 and Rs4231 (75.9–76.9% amino acid identity), that are able to use the human ACE2 receptor for cell entry11 (Supplementary Table 7). In addition, the RBD of the spike protein from WHCV was only one amino acid longer than the RBD of the spike protein from SARS-CoV (Extended Data Fig. 7a). By contrast, other bat SARS-like CoVs—including the Rp3 strain that cannot bind to human ACE214—had amino acid deletions at positions 433–437 and 460–472 compared with the sequence in SARS-CoVs (Extended Data Fig. 7a). The previously determined15 crystal structure of the RBD of the spike protein of SARS-CoV complexed with human ACE2 (Protein Data Bank (PDB) 2AJF) revealed that regions 433–437 and 460–472 directly interact with human ACE2 and hence may be important in determining species specificity (Extended Data Fig. 7b). We predicted the three-dimensional protein structures of the RBD domains of the spike protein of WHCV, Rs4874 and Rp3 by protein homology modelling using the SWISS-MODEL server and compared them to the crystal structure of RBD domain of the spike protein of SARS-CoV (PDB 2GHV) (Extended Data Fig. 7c–f). In accordance with the sequence alignment, the predicted protein structures of the RBD domains of WHCV and Rs4874 were closely related to that of SARS-CoV and different from the predicted structure of the RBD domain from Rp3. In addition, the N terminus of the spike protein of WHCV is more similar to that of SARS-CoV than other human coronaviruses (HKU1 and OC43) (Extended Data Fig. 8) that can bind to sialic acid16. In summary, the high similarities of the amino acid sequences and predicted protein structures of the RBD domains of WHCV and SARS-CoV suggest that WHCV may efficiently use human ACE2 as a receptor for cellular entry, which could potentially facilitate human-to-human transmission11,17,18.
To further characterize the putative recombination events in the evolutionary history of the sarbecoviruses, the whole-genome sequence of WHCV and four representative coronaviruses—bat SARS-like CoV Rp3, CoVZC45, CoVZXC21 and SARS-CoV Tor2—were analysed using the Recombination Detection Program v.4 (RDP4)19. Although the similarity plots suggested that possible recombination events had occurred between WHCV and SARS-CoVs or SARS-like CoVs (Extended Data Fig. 9), there was no significant evidence for recombination across the genome as a whole. However, some evidence for past recombination was detected in the S gene of WHCV, SARS-CoV and bat SARS-like CoVs (WIV1 and RsSHC014) (P < 3.147 × 10−3 to P < 9.198 × 10−9), for which the similarity plots suggested the presence of recombination breakpoints at nucleotides 1,029 and 1,652, which separate the S gene of WHCV into three regions (Fig. 3). In phylogenies of the nucleotide fragments from 1 to 1,029 and from 1,652 to the end of the sequence, WHCV was most closely related to bat SL-CoVZC45 and bat SL-CoVZXC21, whereas in the region of nucleotides 1,030 to 1,651 (the RBD region) WHCV grouped with SARS-CoV and bat SARS-like CoVs (WIV1 and RsSHC014) that are capable of direct human transmission17,20. Despite these recombination events, which seem relatively common among sarbecoviruses, there is no evidence that recombination has facilitated the emergence of WHCV.
Coronaviruses are associated with a number of infectious disease outbreaks in humans, including SARS in 2002–2003 and Middle East respiratory syndrome (MERS) in 20121,21. Four other coronaviruses—human coronaviruses HKU1, OC43, NL63 and 229E—are also associated with respiratory disease22,23,24,25. Although SARS-like coronaviruses have been widely identified in mammals including bats since 2005 in China10,26,27,28, the exact origin of human-infected coronaviruses remains unclear. Here we describe a new coronavirus—WHCV—in the BALF from a patient who experienced severe respiratory disease in Wuhan, China. Phylogenetic analysis suggests that WHCV is a member of the genus Betacoronavirus (subgenus Sarbecovirus) that has some genomic and phylogenetic similarities to SARS-CoV1, particularly in the RBD of the spike protein. These genomic and clinical similarities to SARS, as well as its high abundance in clinical samples, provides evidence for an association between WHCV and the ongoing outbreak of respiratory disease in Wuhan and across the world. Although the isolation of the virus from only a single patient is not sufficient to conclude that it caused these respiratory symptoms, our findings have been independently corroborated in further patients in a separate study29.
The identification of multiple SARS-like CoVs in bats have led to the idea that these animals act as hosts of a natural reservoir of these viruses22,23. Although SARS-like viruses have been identified widely in bats in China, viruses identical to SARS-CoV have not yet been documented. Notably, WHCV is most closely related to bat coronaviruses, and shows 100% amino acid similarity to bat SL-CoVZC45 in the nsp7 and E proteins (Supplementary Table 3). Thus, these data suggest that bats are a possible host for the viral reservoir of WHCV. However, as a variety of animal species were for sale in the market when the disease was first reported, further studies are needed to determine the natural reservoir and any intermediate hosts of WHCV.
Note added in proof: Since this paper was accepted, the ICTV has designated the virus as SARS-CoV-2 (30); in addition, the WHO has released the official name of the disease caused by this virus, which is COVID-19 (31).”