SEA-PHAGES | MRA required information

Link to this post \| posted 31 Oct, 2024 15:52
Tamarah_Adair	I am working on MRAs and these are one of the reviewer's comments: – Illumina platform description and a description of how the reads were quality controlled is missing - Line 46: versions used should be added (Newbler and Consed) - a description of how the genome termini were determined is lacking, This is the paragraph I submitted Genomic DNA was isolated using the Wizard® DNA Clean-Up Kit (Promega) and sequenced by the Pittsburg Bacteriophage Institute using Illumina Sequencing. The NEB Ultra II Library Kit, v3 reagents, was used for each phage with read lengths recorded at 150-base single-end reads. The number of reads and average sequence coverage is recorded in Table 1. Reads were assembled using Newbler (8 ) and Consed (9). Any help is much appreciated. Tammy Edited 31 Oct, 2024 17:01

Link to this post | posted 31 Oct, 2024 15:52

I am working on MRAs and these are one of the reviewer's comments:

– Illumina platform description and a description of how the reads were quality controlled is missing

- Line 46: versions used should be added (Newbler and Consed)

- a description of how the genome termini were determined is lacking,

This is the paragraph I submitted

Genomic DNA was isolated using the Wizard® DNA Clean-Up Kit (Promega) and sequenced by the Pittsburg Bacteriophage Institute using Illumina Sequencing. The NEB Ultra II Library Kit, v3 reagents, was used for each phage with read lengths recorded at 150-base single-end reads. The number of reads and average sequence coverage is recorded in Table 1. Reads were assembled using Newbler (8 ) and Consed (9).

Any help is much appreciated.
Tammy

Edited 31 Oct, 2024 17:01

Link to this post \| posted 31 Oct, 2024 16:58
DanRussell	Hi Tammy, This is a common question. I'm actually working on updating the PhagesDB database to include type of sequencer, but in the meantime here's a shorthand: If your phage was sequenced by Illumina at Pitt before February 2024, it was on an Illumina MiSeq. If it was March 2024 or after, it was on an Illumina NextSeq 1000. Similarly, if your phage was sequenced on the MiSeq, we recommend adding "Raw" to make your sentence "Raw reads were assembled…" because for phages run on the MiSeq, we haven't done any additional trimming or QC. This will change for the NextSeq 1000 genomes, so if your phage genome was sequenced on that, the reads were trimmed and filtered by: cutadapt 4.7 (using the option: –nextseq-trim 30) and skewer 0.2.2 (using the options: -q 20 -Q 30 -n -l 50) Versions of other programs: Newbler 2.9 Consed 29 For genomic termini, it's too complicated to describe without using most of the 500 words of your MRA, so I usually tell people to just say "as previously described" and cite the chapter I wrote about precisely that. https://pubmed.ncbi.nlm.nih.gov/29134591/ Hope that helps! Oh, and don't forget the "h" in Pittsburgh! –Dan Edited 31 Oct, 2024 17:00

Link to this post | posted 31 Oct, 2024 16:58

DanRussell

Hi Tammy,

This is a common question. I'm actually working on updating the PhagesDB database to include type of sequencer, but in the meantime here's a shorthand:

If your phage was sequenced by Illumina at Pitt before February 2024, it was on an Illumina MiSeq. If it was March 2024 or after, it was on an Illumina NextSeq 1000.

Similarly, if your phage was sequenced on the MiSeq, we recommend adding "Raw" to make your sentence "Raw reads were assembled…" because for phages run on the MiSeq, we haven't done any additional trimming or QC. This will change for the NextSeq 1000 genomes, so if your phage genome was sequenced on that, the reads were trimmed and filtered by:

cutadapt 4.7 (using the option: –nextseq-trim 30)
and
skewer 0.2.2 (using the options: -q 20 -Q 30 -n -l 50)

Versions of other programs:
Newbler 2.9
Consed 29

For genomic termini, it's too complicated to describe without using most of the 500 words of your MRA, so I usually tell people to just say "as previously described" and cite the chapter I wrote about precisely that.
https://pubmed.ncbi.nlm.nih.gov/29134591/

Hope that helps!

Oh, and don't forget the "h" in Pittsburgh!

–Dan

Edited 31 Oct, 2024 17:00

Link to this post \| posted 31 Oct, 2024 20:19
Tamarah_Adair	Thank you! I appreciate your quick response.

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