147

Good observation! The 3' poly(A) tail is actually a very common feature of positive-strand RNA viruses, including coronaviruses and picornaviruses. For coronaviruses in particular, we know that the poly(A) tail is required for replication, functioning in conjunction with the 3' untranslated region (UTR) as a cis-acting signal for negative strand synthesis ...


44

That is the correct sequence for 2019-nCov. Coronavirus is of course an RNA virus and in fact, to my knowledge, every RNA virus in Genbank is present as cDNA (AGCT, i.e. thydmine) and not RNA (AGCU, i.e. uracil). The reason is simple, we never sequence directly from RNA because RNA is too unstable and easily degraded by RNase. Instead the genome is reverse ...


31

This question is quite general, so I'm going to attempt to tie it back to bioinformatics. Background The tree for the current coronavirus is here, showing it is closely related to bat-coronavirus and in particular SARS. Question The bioinformatics question for the current coronavirus is why this virus appears to be able to infect humans and transmit to ...


30

The earliest mention of the 30x paradigm I could find is in the original Illumina whole-genome sequencing paper: Bentley, 2008. Specifically, in Figure 5, they show that most SNPs have been found, and that there are few uncovered/uncalled bases by the time you reach 30x: These days, 30x is still a common standard, but large-scale germline sequencing ...


24

The scenarios are impossible and would be laughable if they were not so serious. The evidence is in the phylogenetic trees. Its a bit like a crime scene when the forensics team investigate. We've done enough crime-scenes often going to the site, collecting the pathogen, sequencing and then analysis - (usually neglected diseases) without any associated ...


20

Some of the other answers here seem quite good; at the same time I think the core answer to the OP's question is maybe a bit hard to tease out of them, so I'd like to try to state it more plainly. It's worth noting that a truly complete answer to this question seems to be beyond current research, but any kind of "Why?" is inevitably a hard or even impossible ...


16

The maternal and paternal copies of a chromosome are called haplotypes. Many metazoans (animals) are diploid and have maternal and paternal chromosome contribution during sexual reproduction, not just humans as your question states. Response to Q1 Your question, in other words, is: Why do .bam files not differentiate between haplotypes? Your question ...


16

See IUPAC codes: So, as you can see above, K means "Either G or T".


16

Most sequencing experiments, be it Illumina-based next-generation-sequencing or Sanger sequencing uses DNA as template, not RNA. Even if this virus is RNA-based it would be reverse-transcribed prior to any sequencing experiment. Therefore the output is DNA and this is what NCBI provides here.


15

Actually this is one of the main problems you have when analyzing scRNA-seq data, and there is no established method for dealing with this. Different (dedicated) algorithms deal with it in different ways, but mostly you rely on how good the error modelling of your software is (a great read is the review by Wagner, Regev & Yosef, esp. the section on "...


13

Not an expert, but some searching on eukaryotic positive-strand RNA viruses seems to show that polyadenylation is not uncommon. For example, Steil, et al., 2010.


12

PacBio does use PHRED 33, but it turns out the question may be irrelevant for the newer PacBio Sequel Sequencer, because it reports all base qualities as PHRED 0 (ASCII !). The RS-II reports PHRED 33 quality scores. The Sequel provides data in the form of subreads, which are the circular consensus sequences (CCS) from a single zero-mode waveguide (ZMW). ...


12

In any scenario where depth of coverage is an important factor, PCR duplicates erroneously inflate the coverage and, if not removed, can give the illusion of high confidence when it is not really there. For example, consider the following hypothetical scenario. * ...


11

UPDATE: The article has now been withdrawn with the following note: This paper has been withdrawn by its authors. They intend to revise it in response to comments received from the research community on their technical approach and their interpretation of the results. If you have any questions, please contact the corresponding author. This is very ...


10

I found a post useful for this topic. It explains the difference of coverage and depth. It also has a useful explanation on how to calculate coverage and depth. Here is a copy of what the link says just encase the post is removed: Depth of coverage How strong is a genome "covered" by sequenced fragments (short reads)? Per-base coverage is ...


9

There are several questions in your post I'll try to answer each one: Is there any way to calculate how deep the sequencing is ? See gringer's answer. TLDR: The depth of the sequencing is how many times each position has been sequenced. What should be the optimum depth to get reliable data ? The optimum depth depends on what you want to do with that ...


9

Things like this might depend on your specific library prep, but in general: sequencing starts at the end of the adapter, not before it. You will only see adapters if you sequence through the entire fragment into the adapters on the other side. If your fragment is long enough you won't see any adapter, and of course, that's the desirable outcome of your ...


8

Sequencing depth is typically calculated as the number of total bases sequenced divided by the number of bases in the target genome. An Illumina sequencing run with 2x125 bp reads and 500 million read pairs sequenced would be a sequencing depth of about 40X (assuming my calculations are correct for a 3 billion base genome). The sequencing depth depends on ...


8

Short answer: yes, but you need to get permission (and modified software) from ONT before doing that. ... but that doesn't tell the whole story. This question has the potential to be very confusing, and that's through no fault of the questioner. The issue is that for the MinION, sequencing (or more specifically, generating the raw data in the form of an ...


8

tldr - The I*.fastq.gz file contains the read index sequences. long explanation Illumina uses a program called bcl2fastq to demultiplex sequencing runs. This software takes a list of samples and their associated indices and uses those sequences to make one or more fastq files per sample, binned by one or two index sequences on either end of the sequencing ...


7

Solexa Inc. sequenced NA12878 chrX to ~30x in early 2007, which later became part of Bentley (2008). This, I believe, was the first time that 30x showed up. I don't recall they had a particular reason for that. Figure 5 in the published paper was just aftermath. It does not really explain why not 25x or 35x, given that the curves between 25x and 35x in that ...


7

PCR polymerases introduce errors. When an error arises in the first few cycles of amplifications, it will appear in a reasonably high fraction of DNA fragments in the library. After sequencing, you may see the same error occur to multiple reads. If you remove PCR duplicates when calling variants, all errors are reduced down to one read. For high-coverage ...


7

There are also third party free and open source basecallers that haven't been developed by Oxford Nanopore. Of particular note is Chiron, which gave the best uncorrected assembly identity among the base callers that Ryan Wick tested. It's slower than Albacore, but appears to be more customisable, and could theoretically be used to model any DNA feature that ...


7

Insertions and deletions are the dominant error mode of long read sequencing, including nanopore sequencing. What you see is not unexpected. Things may have improved by now if you would download the raw fast5 data and repeat the basecalling. There is no need to gunzip the fastq.gz prior to alignment. Your commands for alignment look alright, except that (if ...


7

Normally "inserts" used in the manuscript are "indels" in protein alignments, short for insertions and deletions. What I think has happened is a group investigating indels in HIV env noticed indels in 2019-nCov. Essentially I think the correlation is spurious - but I haven't test it, but the area of research in understanding indels is certainly valid and ...


6

As asked as it is, the answer is probably no. Indeed, the highest expressed RPKM/FPKMs will be different from one condition (or tissue) to another one for example. Then, you may also have technical artefacts due to the wet-lab part or to the normalisation. For example, mitochondrial genes are often reported in the top expressed genes. Now, if you want to ...


6

30 times coverage is not unique to this problem, but number 30 has its empirical role in statistics: In statistical analysis, the rule of three states that if a certain event did not occur in a sample with n subjects, the interval from 0 to 3/n is a 95% confidence interval for the rate of occurrences in the population. When n is greater than 30, this is a ...


6

Yes, you can click "stop acquisition" and your run won't be negatively affected. All of the reads are saved as they are generated. I am not sure how this will impact live basecalling though if that is something that you do.


6

Yes, your understanding is largely correct. This originates from the situation that for each detector on a nanopore array there are 4 pores. I'll explain mux scans and groups, but this is outdated information as now another system is used. So a MinION FC has 2048 pores with 512 sensors. Not all of these pores will be equally suitable for sequencing. At the ...


6

Bits are not frequencies. If a position only contains an A (position 3 for example) then you would need 2 questions (bits) to derive that information without a priori knowledge. Is it a G or C? if no > then it is a A or T Is it T? If no then it is an A Position 1 can be derived by 1 question only: is it a G or C? If no then it is an A or T In this case ...


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