Do we have any information on how the hit calls were made? The only detail I see in the data description is we used 3 rounds of selection in triplicate to identify binders experimentally.
Does this mean that hits were called purely off PCR counts after 3 rounds of selection? If so, what was the process of deciding the cutoff? Was there any post-screen hit validation?
", "messages": [ { "id": 2854032, "postDate": "2024-06-04T05:11:08.097Z", "content": "Do we have any information on how the hit calls were made? The only detail I see in the data description is we used 3 rounds of selection in triplicate to identify binders experimentally.
Does this mean that hits were called purely off PCR counts after 3 rounds of selection? If so, what was the process of deciding the cutoff? Was there any post-screen hit validation?
", "rawMarkdown": "Do we have any information on how the hit calls were made? The only detail I see in the data description is `we used 3 rounds of selection in triplicate to identify binders experimentally`.\n\nDoes this mean that hits were called purely off PCR counts after 3 rounds of selection? If so, what was the process of deciding the cutoff? Was there any post-screen hit validation?", "votes": 6 }, { "id": 2854443, "postDate": "2024-06-04T10:18:35.873Z", "content": "From the Overview tab of this competition:
\n\"These barcoded small molecules are in a pool (many in a single tube, rather than one tube per small molecule) and are exposed to the protein target of interest in solution. The protein target of interest is then rinsed to remove small molecules in the DEL that don’t bind the target, and the remaining binders are collected and their DNA sequenced.\"
", "rawMarkdown": "From the Overview tab of this competition:\n\n\"These barcoded small molecules are in a pool (many in a single tube, rather than one tube per small molecule) and are exposed to the protein target of interest in solution. The protein target of interest is then rinsed to remove small molecules in the DEL that don’t bind the target, and the remaining binders are collected and their DNA sequenced.\"", "replies": [ { "id": 2855290, "postDate": "2024-06-04T17:42:17.030Z", "content": "That's an overview of how DEL screening works but that doesn't give any information about how hits are called.
\nAfter each round of selection, you elute your ligand pool from your protein and PCR amplify the barcodes in the post-selection pool. Then you sequence the PCR results. The literal output of the assay is count data for different DNA barcodes.
\nBarcode counts on their own are not hit calls. There is a distribution of counts in the final pool where a small number of ligands have high counts and a large number of ligands have low counts. For an example, see Figure 8 here. Generally you would not assume that everything in the final pool is a hit.
\nTypically a person analyzes the count data and decided a reasonable cutoff for what is considered a hit. That is the process I'm asking about - I'd like to know more about how the raw outputs of the DEL screen were resolved into the competition dataset.
", "rawMarkdown": "That's an overview of how DEL screening works but that doesn't give any information about how hits are called. \n\nAfter each round of selection, you elute your ligand pool from your protein and PCR amplify the barcodes in the post-selection pool. Then you sequence the PCR results. The literal output of the assay is count data for different DNA barcodes.\n\nBarcode counts on their own are not hit calls. There is a distribution of counts in the final pool where a small number of ligands have high counts and a large number of ligands have low counts. For an example, see Figure 8 [here](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6080696/). Generally you would _not_ assume that everything in the final pool is a hit.\n\nTypically a person analyzes the count data and decided a reasonable cutoff for what is considered a hit. That is the process I'm asking about - I'd like to know more about how the raw outputs of the DEL screen were resolved into the competition dataset.", "votes": 4 } ] }, { "id": 2857433, "postDate": "2024-06-05T21:36:31.623Z", "rawMarkdown": "", "isDeleted": true } ], "comments": [ { "id": 2854443, "author_name": "John Mitchell", "author_url": "", "post_date": "2024-06-04T10:18:35.873000", "content": "From the Overview tab of this competition:
\n\"These barcoded small molecules are in a pool (many in a single tube, rather than one tube per small molecule) and are exposed to the protein target of interest in solution. The protein target of interest is then rinsed to remove small molecules in the DEL that don’t bind the target, and the remaining binders are collected and their DNA sequenced.\"
", "votes": 0, "replies": [ { "id": 2855290, "author_name": "Karl Heyer", "author_url": "", "post_date": "2024-06-04T17:42:17.030000", "content": "That's an overview of how DEL screening works but that doesn't give any information about how hits are called.
\nAfter each round of selection, you elute your ligand pool from your protein and PCR amplify the barcodes in the post-selection pool. Then you sequence the PCR results. The literal output of the assay is count data for different DNA barcodes.
\nBarcode counts on their own are not hit calls. There is a distribution of counts in the final pool where a small number of ligands have high counts and a large number of ligands have low counts. For an example, see Figure 8 here. Generally you would not assume that everything in the final pool is a hit.
\nTypically a person analyzes the count data and decided a reasonable cutoff for what is considered a hit. That is the process I'm asking about - I'd like to know more about how the raw outputs of the DEL screen were resolved into the competition dataset.
", "votes": 4, "replies": [] } ] }, { "id": 2857433, "author_name": "", "author_url": "", "post_date": "2024-06-05T21:36:31.623000", "content": "", "votes": 0, "replies": [] } ], "raw_markdown_by_id": { "2854032": "Do we have any information on how the hit calls were made? The only detail I see in the data description is `we used 3 rounds of selection in triplicate to identify binders experimentally`.\n\nDoes this mean that hits were called purely off PCR counts after 3 rounds of selection? If so, what was the process of deciding the cutoff? Was there any post-screen hit validation?", "2854443": "From the Overview tab of this competition:\n\n\"These barcoded small molecules are in a pool (many in a single tube, rather than one tube per small molecule) and are exposed to the protein target of interest in solution. The protein target of interest is then rinsed to remove small molecules in the DEL that don’t bind the target, and the remaining binders are collected and their DNA sequenced.\"", "2857433": "" } }