Table 5 Quality Control event descriptions, triggers, actions, and notifications

A

Poor quality

Warn

Casper mismatch filter > 95% PNN data

In sample X, the Casper alignment step discarded high level of pairs due to mismatches.

Please ensure the quality of amplification reagents not compromised (check polymerase expiration date, proper storage conditions, quality of primers). Please confirm the concentration and quality of pooled library and ensure the correct concentration loaded on the chip. Check the expiration date on the MiSeq reagent kit.

B

Poor purification

Warn

Primer dimer filter > 95% PNN data

In sample X, the primer dimer filter shows a high level of pairs discarded

Please check the concentration of primers (barcode and index) and review magnetic beads cleaning procedure. Ensure the quality of your final pooled library exceeds 95% purity.

C

Negative or loss of product detection

Reject, warn

Primer dimer filter > 0.785 or minority MID filter > 0.11

Sample X was determined to be either a negative control or suffered a loss of product during library preparation.

If this was not intended to be a negative control, please check samples proximity to an intended negative control, and if the negative control passes, consider that there may have been mislaballing. Please repeat the library preparation for this sample.

D

Unclassified sequences form dominant population for sample

Warn, notify CDC

Proportion of sequences that cannot be classified is higher than for any other subtype.

Sample X cannot be classified. CDC/DVH has been notified.

Please wait to be contacted by CDC staff.

E

Subtype classification issues

Warn

Sequences within the population has a best subtype match and second best subtype match with ratio < 2

For sample X, ambiguous subtype classifications have been detected.

Please note that this sample’s subtype is questionable.

F

Chordless cycle detected

Warn, notify CDC

Analysis task contains a chordless cycle of 4 nodes or more

Chordless cycle detected in samples X, Y, and Z.

Please check for signs of contamination between samples X, Y, and Z, and repeat library if feasible.

G

Residual read-pair level too low

Reject

Read pair count < 10,000 after all other filters execute

Sample X does not have enough reads after all filters execute to proceed.

Please review the sample preparation for sample X. Repeat this sample in next library if feasible. If not, contact ghost@cdc.gov about relaxing read pair level restrictions.