The following description is for samples that are decided to be processed by pooling.

1. Use the information in the case sheets to prepare an Excel spreadsheet that would contain the information for each sample provided in the case sheets. This is your "Sample Sheet" for a given Lot.
2. Group these samples into separate hospitals/districts for every day separately, with the date and time of sample reception. Please use separate registers/notebooks for each hospital/district if you are not using computerized system. Label such groups as "Lot-1", "Lot-2", and so on. Within each "Lot", create bunch of five vials (picked randomly from the Lot) and label them as "Pool-1", "Pool-2", and so on.
3. Pipette 30 µl of each sample (from a pool of 5) into a common 1.5 ml microcentrifuge tube to make a total of 150 µl. This tube has same label as that of respective pool. If the sample numbers are not in multiples of 5, combine 30 µl from each of the last 1/2/3/4 samples in the last pool and adjust the volume to 150 µl using 1x PBS.
4. Immediately after completing one pool, secure the 5 tubes with a rubber band or put in a plastic beaker and label the pool. Ensure each pool stays as a discrete entity. Place multiple such pools in cardboard boxes and store them at 4°C fridge.
5. Add lysis buffer to 150 µl of pooled samples.
6. RNA isolation, reverse transcription, and real-time PCR: These steps proceed as usual, without any major changes because of the pooling. Elute in lesser volume (35 µl) to concentrate RNA.The Ct value cut-offs to classify a pool as negative or positive will need to be increased by 3-4 cycles due to dilution during pooling to ensure that sensitivity is not compromised.
7. Once all qRT-PCRs are done, the pools that tested positive will be re-analysed by preparing RNA from individual samples of only those pools.

1. Make a list of the unique identifiers of the samples in the pools that tested positive.
2. Carry printouts of "Sample Sheet", the alphabetically-sorted patient details, of all the separate "Lots" into BSL-II/III lab.
3. Take out only the pools that tested +ve from the fridge and now assign individual unique identifiers to each of those tubes, by marking on the printed list.
4. Label the tubes and proceed with aliquoting and lysis as usual.
5. Proceed with RNA isolation, reverse transcription and real-time PCR as usual.
6. Store all the negative pools (with the existing rubber bands and labels) in a biohazard bag and keep inside BSL II/III lab until disinfected/autoclaved and discarded, as per the guidelines provided by the competent authority.

All individual samples that belonged to the negative pools will be reported as negative for SARS-CoV-2. The respective pool data (Ct value, etc.) will be associated with the five negative samples. Every single sample from the positive "pools" after re-testing individually will be reported as positive/negative, accordingly, with associated data (Ct value, etc.)