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What is the preferred method of pooling BACs for sequencing using 454? We are pooling 48 BACs across 4 lanes. We have an idea of the individual BAC sizes (and they differ considerably). Is it better to have like sizes together in the same lane, or rather ensure that the average KB size is similar for each of the 4 lanes.
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Good question, I'll be watching for an informed answer... (I'm a noob).The king of reverse takeovers, get the scoop here. -
Are you sure you are doing 454 sequencing? The term "lane" is usually reserved for Illumina sequencing.
The most important thing is to ensure you don't pool any two BACs which could be similar at the DNA level! Otherwise you won't be able to untangle them in the de novo assembly stage.Comment
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Thanks for your reply Torst. I am very new to NGS so am not yet "fluent in the lingo"... Your are correct re the lanes, what I meant is that a bunch of BACs are being pooled in 1/4 lane - we will be doing a whole plate though. All individual BACs will be tagged so will be able to determine which BAC the reads originated from.
What I am trying to understand is whether there is a logical way of pooling BACs to ensure that there is little bias in coverage of individual BACs. ie if we have a BAC that is 50kb in the same pool as one that is 150kb, should we adjust the amount of either BAC within the pool? Otherwise is it a better idea to sort all the BACs on size and put 'like sizes' into the same pool?
This might be a petty adjustment and not require a second thought, just wanted to hear from the experts...Comment
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I haven't done this, but trying to balance the pools by BAC size (including the vector) would seem like the way to go. That would balance your expected number of reads for each BAC.Comment
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We can assume there is a fixed number of reads available to each pool ie. a 1/4 plate worth of reads.
Within a pool, the number of reads a particular BAC gets is proportional to its size.
So, as Keith says, you would probably want to ensure that the sum of BAC sizes in each pool was similar between pools.
(Sorry I missed that each BAC is tagged/barcoded - in that case my response about keeping similar-DNA BACs separate doesn't matter.)
So I guess you are using four pools instead of one is so you can reuse the tags/barcodes in each pool?Comment
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...
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