I need to know that how can ve decide that this sampke needs 1 million reads so we have to load this much sample, where the otherone sampke needs 200k reads then it would be loaded the xyz.
You need to assess the throughput of the sequencing technology you use. As an example, if you are using a sequencer with an average total output of 2,000,000 reads, then you would split your pooling in such a way that your larger sample would generate at least one million reads, and your smaller sample would generate 200K reads. If you needed one million reads per sample, you would pool in an even split (1:1). However, in this case, as your sequencer will generate more reads than you need, you can split 2:1 which would give you 660Kish reads for the smaller sample and 1.3ish million reads for the larger sample. This is all assuming that the library sizes are the same. If the library sizes are NOT the same, then you would have to factor that in as well (e.g a smaller library would have a higher coverage with the same amount of reads).
Always account for pipetting or quantitation errors, and aim for slightly more reads than you actually need so that you meet your minimum output threshold.
Thank yousophie sneddon.
Can you make me more clear for the Gene studio S5 sequencer machine we have and we are going to do the NIPT test by using 540 chip. manufacturer saying that this chip has potential to genwrate 80 million reads. so do you suggest how many samples should we pool in single chip..???
Do you working on diagnosis or in research...???
I work in research. If the manufacturer is saying you will generate 80 million reads, then theoretically you could pool 80 samples at an equal mass each and generate 1 million reads per sample. I would underestimate that value though, as you will never hit the maximum throughput (but you will likely come close unless something goes very wrong). Not all reads will reach the quality threshold and will be filtered from analysis.
I am not familiar with the Gene Studio SS5 sequencing technology. I work primarily with Illumina technology, and have worked with Ion Torrent Proton and PGM previously. It looks like it is built to run targeted sequencing experiments, so calculate the size of your library design, and calculate your pooling depending on how many reads you need per sample.
If your library design is small (which it likely is), then you could easily pool your two samples equally and generate far more than 1 million reads and 200K reads per sample.
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