RFI-Amendment 01.docx

Request for Information

Question: Phix during sequencing ok?

Answer: Yes, Phix during sequencing is okay.

Question: Unique alignments: We can do it but Duplicate removal is not recommended for ddRAD as the enzyme used in experiments cuts at specific positions in the genome, and thus library in enriched for those specific fragments causing a very high duplicate rate. Removing duplicates could greatly reduce effective coverage for variant detection.

Answer: If duplicate removal is not recommended for ddRAD and will result in greatly reduced effective coverage for variant detection then this step is not necessary. It is okay to proceed without duplicate removal.

Question: For homozygous SNPs: The most common allele must be supported by at least 80% of all the aligned reads covering that position. At least 5 unique reads must support the most common allele: This can be achieved by applying a variant allele frequency and depth filter post-variant detection. Please confirm that they want to perform this filtering for individual sample levels and not as a whole population.

Answer: We confirm that we want to perform this filtering for individual sample levels and not as a whole population.

Question: Polymorphisms in the first and last 3 base pairs of each read are to be ignored, and each polymorphic base must have at least a PHRED base quality value of 20. The only way we can do this is by trimming 3bp from both ends.

Answer: If erroneous variants are often detected in either the first or last 3 base pairs of reads then this is necessary to do. If erroneous variants are not often detected in the first or last 3 base pairs of reads then this is not necessary.

Question: For heterozygous SNPs: Each of the two most common alleles must be supported by at least 30% of all aligned reads covering that position, at least 5 unique reads must support each of the two most common alleles, the sum of reads of the two most common alleles must account for at least 80% of all aligned reads covering that nucleotide position. This can be achieved by applying a variant allele frequency and depth filter post-variant detection. Please confirm that they want to perform this filtering for individual samples levels and not as a whole population. We might need help understanding this.

Answer: We confirm that we want to perform this filtering for individual sample levels and not as a whole population.

Question: A range of 2,000 – 10,000 markers shall be reported in vcf file format. The only way we can do this is by trimming 3pb from both ends.

Answer: We are confused about this part and believe it may have been a typo. If incorrect, please let us know.

Question: What is the timeline i.e., when would the project start (i.e. samples submitted)?

Answer: As soon as possible. We have lyophilized tissue in Qiagen Catalog #19560 or Corning Life Sciences #4412 plates that we are ready to send as soon as a vendor is identified. We prefer to have the vendor extract the DNA, but if our plates will not fit with their equipment or system, we can extract DNA on site, but would expect the bid price to be reflective of that.

Question: Are other sequencing instruments (e.g. Illumina NovaSeq, or Element Biosciences AVITI) acceptable as “similar” to the Ion Proton? (as you mention “a Life Technologies’ Ion Proton System, or similar”) Answer: Yes, other sequencing instruments, such as those listed, are acceptable as “similar” to the Ion Proton.

Question: Out of curiosity, if using PstI+MspI enzyme combination was recommended to obtain more SNPs in coding regions instead of NspI and BfuCI enzymes, would this work for you all? If not, that is okay.

Answer: Yes, using PstI+MspI enzyme combination to obtain more SNPs in coding regions instead of NspI and BfuCI enzymes would be okay.