This will take a little explanation. I am preparing custom libraries using the following procedure, which is very similar to the Illumina TruSeq procedure with some minor modifications (aka transposon insertion sequencing):
1. Shear DNA to average 300-bp on a Covaris
2. End repair and A-tail
3. Adapter ligation using custom-synthesized indexed adapters that have the same sequence as in the TruSeq kit, and with another adapter that is only the fragment of the universal adapter that is complementary to the indexed adapter
4. Gel extract products in 250-500 bp range or so (there was a little issue at my workplace when doing this, in that our TAE buffer was far too dilute resulting in a smeary run, so it is possible there is still some adapter present in the samples)
5. PCR enrich with a biotinylated forward primer that is specific to the end of a transposon to pull out transposon-chromosome junctions, and contains the full universal adapter sequence as 5' overhang to create it, plus a reverse primer that binds at the end of the indexed adapter. I have some evidence that this PCR reaction worked as expected, and there was hardly any product visible in the gel above 500-bp.
6. Gel extract products in 200-400-bp range. Biotinylated products should probably run a little slower than non-biotinylated, but probably not enough to matter here.
7. Affinity capture biotinylated PCR products on streptavidin beads, and elute off the non-biotinylated strand.
8. Quantify the single-stranded library with KAPA qPCR kit.
The qPCR appeared worked well and I have amplification curves with a 1:100 dilution that look like the samples are between 1-5 nM. However, I ran the qPCR products on a gel just to see what they look like, and they appear to be centered in the 600-700 bp region. I am trying to figure out how this is possible - if it is just an artifact of the 35 cycles of PCR or if this is actually related to the size distribution of my library? I need to estimate an average size of the library fragments to be able to accurately quantify, and just have no idea what this is! The qPCR DNA standard is 452-bp and ran exactly where it was supposed to. Thanks for any suggestions.
1. Shear DNA to average 300-bp on a Covaris
2. End repair and A-tail
3. Adapter ligation using custom-synthesized indexed adapters that have the same sequence as in the TruSeq kit, and with another adapter that is only the fragment of the universal adapter that is complementary to the indexed adapter
4. Gel extract products in 250-500 bp range or so (there was a little issue at my workplace when doing this, in that our TAE buffer was far too dilute resulting in a smeary run, so it is possible there is still some adapter present in the samples)
5. PCR enrich with a biotinylated forward primer that is specific to the end of a transposon to pull out transposon-chromosome junctions, and contains the full universal adapter sequence as 5' overhang to create it, plus a reverse primer that binds at the end of the indexed adapter. I have some evidence that this PCR reaction worked as expected, and there was hardly any product visible in the gel above 500-bp.
6. Gel extract products in 200-400-bp range. Biotinylated products should probably run a little slower than non-biotinylated, but probably not enough to matter here.
7. Affinity capture biotinylated PCR products on streptavidin beads, and elute off the non-biotinylated strand.
8. Quantify the single-stranded library with KAPA qPCR kit.
The qPCR appeared worked well and I have amplification curves with a 1:100 dilution that look like the samples are between 1-5 nM. However, I ran the qPCR products on a gel just to see what they look like, and they appear to be centered in the 600-700 bp region. I am trying to figure out how this is possible - if it is just an artifact of the 35 cycles of PCR or if this is actually related to the size distribution of my library? I need to estimate an average size of the library fragments to be able to accurately quantify, and just have no idea what this is! The qPCR DNA standard is 452-bp and ran exactly where it was supposed to. Thanks for any suggestions.
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