Q--MISEQ DNA SEQUENCING 2 X 300, PER RUN

For a vendor to be considered for award all amendments must be signed and returned with their bid This amendment is being issued to extend the close date from September 5, 12:00 Noon ET to September, 7 1:00 PM ET. Vendors have had many questions This amendment is being issued to 1. Extend the close date from September, 5 12:00 Noon ET to September, 7 1:00 PM ET. 2. To Post answers to vendor questions Can you please clarify the type of amplicons that will be submitted and the fragment size ; a) partial Illumina adapter ligated amplicons - which needs indexing and sequencing b) Illumina adapter and indexed amplicons- this would need only direct sequencing c) raw amplicons- which needs Illumina adapter and indexing (complete library preparation and sequencing) Raw amplicons 2. Assuming raw amplicons will be submitted ¿ TruSeq kit as mentioned allows for only upto 96 indexes. It is mentioned upto 150 samples may be submitted for sequencing on one flow cell. Can you please clarify if the amplicons will have in-line barcodes and the samples will be pooled together? Please elaborate on the sample submission. If in-line barcodes will be present- will in-line barcode de-multiplexing be needed ? along with Illumina index de-multiplexing ? Please let us know. In line barcodes are present, and don¿t need de-multiplexing 3. Can you please comment on the number of batches, samples per batch and the timeline of the sample submission. ¿ we would need this information to accurately provide you with a quote. Batches are up to 4 in the course on contract, with up to 6 runs, some some batches may have 2-3 runs. Anticipate first batch in fall of 2017 and the final batch in late summer of 2018. Timing of intermediate batchesis unknown. "1) What actually is being provided to the vendor? Is it extracted DNA? Amplified DNA, but DNA that is not indexed? DNA that is amplified and indexed? It is unclear, because it sounds like the DNA has already been amplified and indexed, but the offeror is stating that library prep is needed. Library prep is only needed if the DNA has not been amplified and indexed (or at least indexed). 2) I am not sure that the quality control requirements are reasonable. We have used the v3 2x300 kits before. There is no chance of getting 70% of the bases >Q30. These kits just don¿t work well on the Miseq¿s. Also you won¿t get 40M reads passing filter for an individual run. Also note that many of your amplicons will exceed 600 bp which makes getting merged reads impossible for those. You might want to adjust your requirements here, or switch to a 2x250 kit, which we have found works much better.' Using a dual-index sequencing strategy GLKN will produce PCR DNA amplicons from filtered water and tissues using metagenomics primer sets, combined with an Illumina adaptor, 8 bp index sequence, 10 bp pad sequence and 2 bp linker sequence. Amplicon length of the provided DNA fragments will be 400-650 bp. In the provided samples, the amplicons will be barcoded and pooled in equal concentration for sequencing. Yet, the next statement highlighted below indicates that the vendor is expected to prepare libraries using the Illumina TruSeq Kit, which would have already been accomplished per the above statement. The vendor will perform library preparation using the Illumina TruSeq Kits, or comparable library chemistry, that allows 96 indexes and give comparable output quality to TruSeq. The question is: are the libraries indeed premade and ready to run, or is the vendor expected to prepare the libraries from the amplicon material?" Vendor must prepare library from `raw¿ amplicons, that will have a barcode index incorporated. No Illumina indexes are in raw amplicons. What should be included in Technical capability? Examples of problems in projects like this and resolution, Degrees held by staff. For a vendor to be considered for award all amendments must be signed and returned with their bid