This might be a dumb question, but my understanding of biological replicates was that measurements taken from biologically distinct samples under the same experimental conditions(meaning from the same human host). Are isolates the same biological species but from different human hosts? If so why are transcription values across rep1 and rep2 so different?

Created by aark
Hi Monica, Thanks for the question. Isolate 27 was indeed used as batch control, hence why it has multiple BReps. All of the samples in the files provided passed QC. Any samples that failed QC were removed from the data sets. I hope this answers your question. -Sage
Hello @FerdigLab , For SubChallenge 1, there are some isolates with more than 2 Biological replicates in the training data (e.g. isolate 27). Could you please clarify which of these was used as batch control and which one had QC issues? If, for example, isolate 27 didnīt pass QC, should we assume that only some of the Biological replicates (e.g. BRep 7 and BRep8) are of good quality? Thanks. -Monica
Hi wudi, Thanks for the question. For each submission each isolate should only have one prediction. For more information please look at step 4 of this page [https://www.synapse.org/#!Synapse:syn16924919/wiki/590949]. Example file formats can be found here [https://www.synapse.org/#!Synapse:syn18634472]. Thanks. -Sage
@FerdigLab Hi, I have a basic question. For our submission, do we need to make sure all predictions are the same for one isolate? Thanks.
Hi, Some isolates were used as batch controls, i.e., run with every batch regardless, and have more biological replicates. Some isolates were rerun for a third (or more) biological replicate if any of the samples failed QC at a later point. The IC50 value reported in the data set is measured as part of a separate experiment. Briefly, a culture is is exposed to a gradient of drug concentrations for 72 hours starting at early ring stage (0~12 hours post invasion) and the concentration at which 50% of the parasites growth is inhibited. For more information about the IC50 protocol, I recommend [https://www3.nd.edu/~bsmith3/pdf/BMC2017.pdf], specifically section 4.4. Hypoxanthine incorporation growth inhibition assay. Thanks. -Sage
Thanks for the prompt and comprehensive response! It definitely clarified some things. I have a few more questions. Why do some isolates have more than two biological replicates? Is it because the lab just happened to sequence more replicates for just that particular isolates? Also, is the IC50 value reported in the dataset measured 24hrs after the drug has been infused?
Hi, Thanks for the question. My understanding is that there are slight variations in what a "biological replicate" means from field to field. I think a few definitions might be helpful here. Isolate = Malaria parasite collected from a patient. Each isolate is unique and clonal for this challenge. _in vitro_ culturing of malaria = Malaria isolates that have been collected from the field are lab adapted to be grown in a lab. The transcription samples generated for this challenge were all collected from malaria parasites growing in flasks, **with the exception of the sub-challenge 2 training set, which consists of transcription samples of malaria parasites collected straight from the patient**. We provide the malaria parasites with media, gas and human blood (only red blood cells) from a blood donor within the flask. The media and blood donor (or blood batch) may be different from collection to collection. In our field (studying malaria _in vitro_), often times a biological replicate is a second sample collected from the same malaria parasite isolate at different dates, which is the case for this challenge. Because the collection is _in vitro_, each parasite isolate and biological replicate are collected from different flasks, not different human hosts. However, it is highly likely that the blood batch, or the donor that donated the blood, for each biological replicate and isolate, are different. So, the only difference between bio rep 1 and bio rep 2 of any given isolate is the date on which the transcription samples were collected. Then why are the transcription values so different? There are many factors that can affect the transcription of malaria, even across different dates. A huge factor that can affect the transcription of malaria, for example, is developmental stage. Malaria, in the intra-erythrocytic cycle, has a 48 hour life cycle which heavily influences the transcription. It is not uncommon to see slight differences in stage, and hence changes in transcription, between two biological replicates from the same isolate, due to slightly different timings in collection in regards to developmental stage. This is why for sub-challenge 2, the most likely developmental stage, based on transcription, has been provided to the user. We try to control for the stage differences by synchronizing the parasites as best we can. However, no synchronization method is perfect, and inevitably there will be some variation in stage and distribution among all our samples. Another factor that can change from biological replicate to replicate are things such as blood batch and media batch. We try to correct for these as much as possible through things such as batch correction, same media recipe, and always using O+ blood type, but it is possible that there may be some batch affects that have a larger impact on some isolates than others. **To summarize: Biological replicates are the same malaria parasite, collected on different dates. All transcription samples are collected from malaria parasites grown in flasks, with the exception of the sub-challenge 2 training data. Why the transcription values of biological replicates are so different are most likely due to technical differences beyond our control. This is why we provide multiple biological replicates for all isolates across treatments and time points.** Thanks. I hope this answers your question. If you need anything clarified or if you have any more questions, please let us know. -Sage

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