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Hi everyone,
My first questions. I am quite a noob, so please forgive me if I say or ask anything silly. My questions are about contig creation in the paper called 'A new coronavirus associated with human respiratory disease in China', which is here:
https://www.nature.com/articles/s41586-020-2008-3
In the paper, it says this:
"Sequencing reads were first adaptor and quality trimmed using the Trimmomatic program32. The remaining 56,565,928 reads were assembled de novo using both Megahit (v.1.1.3) and Trinity (v.2.5.1) with default parameter settings. Megahit generated a total of 384,096 assembled contigs (size range of 200–30,474 nt), whereas Trinity generated 1,329,960 contigs with a size range of 201–11,760 nt. All of these assembled contigs were compared using BLASTn and Diamond BLASTx) against the entire non-redundant (nr) nucleotide and protein databases."
And this:
"Of the 384,096 contigs assembled by Megahit, the longest (30,474 nucleotides (nt)) had a high abundance and was closely related to a bat SARS-like coronavirus."
So, in summary, after filtering out known human sequences, they were left with 384,096 assembled contigs from Megahit and 1,329,960 assembled contigs from Trinity. And one of these contigs from Megahit was 30,474 nucleotides in length, which was closely related to a bat SARS-like coronavirus.
My questions are:
(a) Are the contigs from Megahit and Trinity individually unique, giving a total of 384,096 + 1,329,960 = 1, 714,056 unique contigs? Or, did Megahit and Trinity both find some of the same contigs, giving a lower number of unique contigs?
(b) Whatever the answer to question (a) above, leaving aside the one 30,474 nucleotide contig which was closely related to a bat SARS-like coronavirus, there remains about a million (or maybe more) contigs of non-human origin in the sample. They are not of human origin, so what is their origin? Obviously there will be other viruses, bacteria, etc, in the bronchoalveolar lavage sample. But a million or more individual unique contigs? I don't know, but that seems like a lot to me. Do these contigs represent a million or more different individual organisms, viruses, bacteria, etc? Or, if not individual organisms, how many different kinds of virus, bacteria, etc, would normally be present in a bronchoalveolar lavage sample? Wow, That question went on a bit longer than I intended. Anyway, please just answer what you can. I don’t expect this question to be answered completely. Maybe some of the things I asked are not actually known.
(c) This question, however, I think may be able to be answered completely. Is it possible that some (or maybe lots) of these million or more contigs could be generated simply by the computer algorithms, and do not actually exist in reality? Because if you have 56,565,928 reads, and only four letters (ATCG) to form the nucleotide strings from, I assume that you would get some overlapping strings in the alignment process, just from the theory of probability alone. Am I correct in assuming this?
(d) If there was a high abundance of strings of 30,000+ nucleotides in the bronchoalveolar lavage sample, wouldn't you be able to find those strings using gel electrophoresis?
I think that’s enough for now. I expect you are all busy people and I don’t want to take up too much of your time. Thank you for reading my post. Any answers I receive will be greatly appreciated.
Warm regards,
Bobby.
My first questions. I am quite a noob, so please forgive me if I say or ask anything silly. My questions are about contig creation in the paper called 'A new coronavirus associated with human respiratory disease in China', which is here:
https://www.nature.com/articles/s41586-020-2008-3
In the paper, it says this:
"Sequencing reads were first adaptor and quality trimmed using the Trimmomatic program32. The remaining 56,565,928 reads were assembled de novo using both Megahit (v.1.1.3) and Trinity (v.2.5.1) with default parameter settings. Megahit generated a total of 384,096 assembled contigs (size range of 200–30,474 nt), whereas Trinity generated 1,329,960 contigs with a size range of 201–11,760 nt. All of these assembled contigs were compared using BLASTn and Diamond BLASTx) against the entire non-redundant (nr) nucleotide and protein databases."
And this:
"Of the 384,096 contigs assembled by Megahit, the longest (30,474 nucleotides (nt)) had a high abundance and was closely related to a bat SARS-like coronavirus."
So, in summary, after filtering out known human sequences, they were left with 384,096 assembled contigs from Megahit and 1,329,960 assembled contigs from Trinity. And one of these contigs from Megahit was 30,474 nucleotides in length, which was closely related to a bat SARS-like coronavirus.
My questions are:
(a) Are the contigs from Megahit and Trinity individually unique, giving a total of 384,096 + 1,329,960 = 1, 714,056 unique contigs? Or, did Megahit and Trinity both find some of the same contigs, giving a lower number of unique contigs?
(b) Whatever the answer to question (a) above, leaving aside the one 30,474 nucleotide contig which was closely related to a bat SARS-like coronavirus, there remains about a million (or maybe more) contigs of non-human origin in the sample. They are not of human origin, so what is their origin? Obviously there will be other viruses, bacteria, etc, in the bronchoalveolar lavage sample. But a million or more individual unique contigs? I don't know, but that seems like a lot to me. Do these contigs represent a million or more different individual organisms, viruses, bacteria, etc? Or, if not individual organisms, how many different kinds of virus, bacteria, etc, would normally be present in a bronchoalveolar lavage sample? Wow, That question went on a bit longer than I intended. Anyway, please just answer what you can. I don’t expect this question to be answered completely. Maybe some of the things I asked are not actually known.
(c) This question, however, I think may be able to be answered completely. Is it possible that some (or maybe lots) of these million or more contigs could be generated simply by the computer algorithms, and do not actually exist in reality? Because if you have 56,565,928 reads, and only four letters (ATCG) to form the nucleotide strings from, I assume that you would get some overlapping strings in the alignment process, just from the theory of probability alone. Am I correct in assuming this?
(d) If there was a high abundance of strings of 30,000+ nucleotides in the bronchoalveolar lavage sample, wouldn't you be able to find those strings using gel electrophoresis?
I think that’s enough for now. I expect you are all busy people and I don’t want to take up too much of your time. Thank you for reading my post. Any answers I receive will be greatly appreciated.
Warm regards,
Bobby.
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