Hi All,
I am pretty new to sequencing on the Miseq, so just learning the ropes with amounts and cluster generation on the flow cell.
I am currently doing ChIP-Seq Experiments and multiplex my libraries by using a 7bp in-line barcode. The way I generate sequence complexity is by multiplexing at least 6 experiments together and spiking 10% PhiX, or so I thought.
I quantified my libraries using KAPA qPCR, and estimated size distribution using Agilent Bioanalyzer 2100 (DNA High Sensitivity). Just being conservative for an initial run I diluted each different Library separately (since amounts were less for some) to 12pM, and since I was adding equal amounts of the library, I mixed equal volumes of each for the final loading volume. I imagine the final concentration should remain the same although each individual library should get diluted. So to 900ul of this combined sample library I added 100ul of 12.5pM PhiX as spike in.
I have attached some of the SAV pics and Run stats as a ppt file for reference.
What I basically observed was that Read1 was really good quality and I got great clusters (98.6% greater than Q30) at a cluster density of 373K/mm2 (Lower than I thought). But as soon as the paired end clusters were formed the quality (% greater than Q30) dropped quite significantly (by 20%). The % aligned reads of the PhiX library drops from 18% in read 1 to ~2% in Read 2. The most bizarre observation was that when the sequence starts reading the sample after the barcode, Read 1 shows a good constant AT:GC ratio, as one would expect, but read 2, some how has a weird C bias. Has anyone encountered this before. Is the run OK, or should I just consider the data from Read 1.
Please provide me your input, as I am thoroughly confused by these Read 2 metrics.
Thanks.
I am pretty new to sequencing on the Miseq, so just learning the ropes with amounts and cluster generation on the flow cell.
I am currently doing ChIP-Seq Experiments and multiplex my libraries by using a 7bp in-line barcode. The way I generate sequence complexity is by multiplexing at least 6 experiments together and spiking 10% PhiX, or so I thought.
I quantified my libraries using KAPA qPCR, and estimated size distribution using Agilent Bioanalyzer 2100 (DNA High Sensitivity). Just being conservative for an initial run I diluted each different Library separately (since amounts were less for some) to 12pM, and since I was adding equal amounts of the library, I mixed equal volumes of each for the final loading volume. I imagine the final concentration should remain the same although each individual library should get diluted. So to 900ul of this combined sample library I added 100ul of 12.5pM PhiX as spike in.
I have attached some of the SAV pics and Run stats as a ppt file for reference.
What I basically observed was that Read1 was really good quality and I got great clusters (98.6% greater than Q30) at a cluster density of 373K/mm2 (Lower than I thought). But as soon as the paired end clusters were formed the quality (% greater than Q30) dropped quite significantly (by 20%). The % aligned reads of the PhiX library drops from 18% in read 1 to ~2% in Read 2. The most bizarre observation was that when the sequence starts reading the sample after the barcode, Read 1 shows a good constant AT:GC ratio, as one would expect, but read 2, some how has a weird C bias. Has anyone encountered this before. Is the run OK, or should I just consider the data from Read 1.
Please provide me your input, as I am thoroughly confused by these Read 2 metrics.
Thanks.
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