Questions and Answers.pdf

Q: The project �tle is “Shotgun” (whole genome) Sequencing, and the Statement of Work sec�ons 1.0 and 2.0 state “to conduct shotgun metagenomic sequencing... However, the Statement of Work Sec�on

3.0 states “Conduct “amplicon metagenomic” sequencing using the NovaSeq pla�orm and goes on to describe an amplicon workflow. Note that the bioinforma�cs required cannot be performed for amplicon sequencing and would require shotgun / WGS. Please confirm that shotgun / WGS is the correct scope of work.

A: The terms amplicon should not have been there and have been removed. It should be shotgun whole genome sequencing.

Q: Regarding bioinforma�cs, SeqMa�c has the ability to develop and/or provide the bioinforma�cs pipeline as writen, with the excep�on of the “Azenta/ GENEWIZ dynamic explora�on of microbial taxonomy and abundance… etc.”. Note that Azenta/GENEWIZ is a direct compe�tor/supplier so no other supplier would know the detailed scope of an Azenta/GENEWIZ offering. As such, please provide a defini�on of the deliverable(s) within the “Azenta/ GENEWIZ dynamic explora�on of microbial taxonomy and abundance…/ pan metagenome” so we have a clear understanding of the scope required.

A: Azenta is willing to perform the library prepara�on, shotgun genome sequencing and then assemble the closed genomes of the fly host, as well as the associated viruses, fungi, and bacteria within the fly. This will be done by taking the reads from all flies sampled and crea�ng a “pan-genome” dra� assembly. They will then use these dra� assemblies as “bins” from which to create the genome assemblies from each fly. This is a necessary step to account for microdiversity between samples. They will then annotate the gene�c content of each microbial genome from the samples and provide full genome feature files. At minimum, the vendor will provide the FASTQ and FASTA files from the sequencing, fully annotated genomes with genome feature files, as well as De Bruijin assemblies at different k-mer lengths.

In addi�on to this, they will include taxonomic analyses which are normally a separate bioinforma�c package. They will pull the full amplicon informa�on from the closed genomes and iden�fy individual amplicon sequence variants (this is why the term amplicon was used previously) for each type of organism (flies, viruses, bacteria, fungi). They will then provide the abundance informa�on and detailed reports including but not limited to NMDS, Krona, Abundance heatmap and Metastats.

Q: We also note comments requiring “gene expression analysis and differen�al expression analysis, etc. (top of page 2), but that is not possible with DNA / shotgun and would require RNA sequencing. Are both DNA and RNA sequencing required in this scope of work?

A: No, it should be just DNA sequencing. Expression statement removed.

Q: Per 3.0: Are you looking for two different quota�ons, i.e.

- one for Metagenomic Sequencing of 1-80 samples, including the bioinformatic analyses

- one for bioinformatics services only - of 1-200 samples? Are the 80 samples for metagenomic sequencing included within the 200 samples for bioinformatics? If the bioinformatics is standalone, exactly what files will be provided to the vendor for bioinformatic services?

A: A single quote combining metagenomic sequencing and bioinforma�cs between 1-200 samples.

Sec�on 3 amended. Files will be generated internally by the servicer.