Hi,
Any tips regarding the use of a custom primer on the NextSeq would be appreciated.
We are trying to run a multiplexed set of 3' RNASeq libraries with a custom sequencing primer on a NextSeq with V2 chemistry with a 75 bp high output kit. The libraries were designed so that all reads come from the 3' UTR just upstream of the poly-A tail. We ran a set of these libs successfully on the MiSeq, but it has failed three times with the NextSeq. The primer contains 18 T's to cover the 18 A's that are at the end of the first strand synthesis primer which is used to make cDNA (see below). We have tried this custom primer with and without the 3' phosphorothioate modification between the final two T's. Without the phosphorothioate we had high phasing (~0.3), low pass filter rates, high error and medium to low intensity, with a total output of 189 M reads. With the phosphorothioate between the last two T's we had very low intensity and only 10 M reads passed filter, but normal cluster density and normal phasing around 0.12. In both cases the barcode reads look excellent and the cell was not over clustered. There were multiple successful runs on this instrument between and after these failed runs.
We were worried that the long stretch of T's might be melting off, but I know the MiSeq runs at 65C while the NextSeq runs at 60 C. So, if melting were the problem then the run should have been worse on the higher temperature of the MiSeq.
The custom primer is underlined here. It largely overlaps with the Illumina read 1 primer:
5’AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-TTTTTTTTTTTTTTTTTT-Insert…
3’TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA-AAAAAAAAAAAAAAAAAA-Insert…
Here are the stats from the latest failed run:
Run Summary
Level Yield Total (G) Projected Total Yield (G) Aligned (%) Error Rate (%) Intensity Cycle 1 % >= Q30
Read 1 11.2 11.2 0 0 3248 79.3
Read 2 0.7 0.7 0 0 5418 96.4
Total 11.85005 11.85005 0 0 4332.993 80.39454
Read 1
Lane Tiles Density (K/mm2) Clusters PF (%) Phas/Prephas (%) Reads (M) Reads PF (M) % >= Q30 Yield(G) Cycles Err Rated Aligned (%) Error Rate (%) Error Rate 35 cycle (%) Error Rate 75 cycle (%) Error Rate 100 cycle (%) Intensity Cycle 1
1 216 210 +/- 61 29.43 +/- 18.55 0.150 / 0.084 136.33 35.55 77.6 3 +/- 0 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 3294 +/- 641
2 216 236 +/- 37 27.98 +/- 20.33 0.089 / 0.052 152.83 40.4 81.4 3 +/- 0 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 2927 +/- 469
3 216 184 +/- 80 29.13 +/- 18.35 0.082 / 0.075 119.12 28.78 79 2 +/- 0 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 3616 +/- 943
4 216 217 +/- 68 22.66 +/- 18.45 0.172 / 0.089 140.47 28.42 78.5 2 +/- 0 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 3154 +/- 1160
Read 2
Lane Tiles Density (K/mm2) Clusters PF (%) Phas/Prephas (%) Reads (M) Reads PF (M) % >= Q30 Yield(G) Cycles Err Rated Aligned (%) Error Rate (%) Error Rate 35 cycle (%) Error Rate 75 cycle (%) Error Rate 100 cycle (%) Intensity Cycle 1
1 216 210 +/- 61 29.43 +/- 18.55 0.000 / 0.000 136.33 35.55 96.2 0 +/- 0 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 4668 +/- 2164
2 216 236 +/- 37 27.98 +/- 20.33 0.000 / 0.000 152.83 40.4 96.8 0 +/- 0 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 5927 +/- 1886
3 216 184 +/- 80 29.13 +/- 18.35 0.000 / 0.000 119.12 28.78 95.7 0 +/- 0 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 5167 +/- 2191
4 216 217 +/- 68 22.66 +/- 18.45 0.000 / 0.000 140.47 28.42 96.9 0 +/- 0 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00
Any tips regarding the use of a custom primer on the NextSeq would be appreciated.
We are trying to run a multiplexed set of 3' RNASeq libraries with a custom sequencing primer on a NextSeq with V2 chemistry with a 75 bp high output kit. The libraries were designed so that all reads come from the 3' UTR just upstream of the poly-A tail. We ran a set of these libs successfully on the MiSeq, but it has failed three times with the NextSeq. The primer contains 18 T's to cover the 18 A's that are at the end of the first strand synthesis primer which is used to make cDNA (see below). We have tried this custom primer with and without the 3' phosphorothioate modification between the final two T's. Without the phosphorothioate we had high phasing (~0.3), low pass filter rates, high error and medium to low intensity, with a total output of 189 M reads. With the phosphorothioate between the last two T's we had very low intensity and only 10 M reads passed filter, but normal cluster density and normal phasing around 0.12. In both cases the barcode reads look excellent and the cell was not over clustered. There were multiple successful runs on this instrument between and after these failed runs.
We were worried that the long stretch of T's might be melting off, but I know the MiSeq runs at 65C while the NextSeq runs at 60 C. So, if melting were the problem then the run should have been worse on the higher temperature of the MiSeq.
The custom primer is underlined here. It largely overlaps with the Illumina read 1 primer:
5’AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-TTTTTTTTTTTTTTTTTT-Insert…
3’TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA-AAAAAAAAAAAAAAAAAA-Insert…
Here are the stats from the latest failed run:
Run Summary
Level Yield Total (G) Projected Total Yield (G) Aligned (%) Error Rate (%) Intensity Cycle 1 % >= Q30
Read 1 11.2 11.2 0 0 3248 79.3
Read 2 0.7 0.7 0 0 5418 96.4
Total 11.85005 11.85005 0 0 4332.993 80.39454
Read 1
Lane Tiles Density (K/mm2) Clusters PF (%) Phas/Prephas (%) Reads (M) Reads PF (M) % >= Q30 Yield(G) Cycles Err Rated Aligned (%) Error Rate (%) Error Rate 35 cycle (%) Error Rate 75 cycle (%) Error Rate 100 cycle (%) Intensity Cycle 1
1 216 210 +/- 61 29.43 +/- 18.55 0.150 / 0.084 136.33 35.55 77.6 3 +/- 0 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 3294 +/- 641
2 216 236 +/- 37 27.98 +/- 20.33 0.089 / 0.052 152.83 40.4 81.4 3 +/- 0 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 2927 +/- 469
3 216 184 +/- 80 29.13 +/- 18.35 0.082 / 0.075 119.12 28.78 79 2 +/- 0 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 3616 +/- 943
4 216 217 +/- 68 22.66 +/- 18.45 0.172 / 0.089 140.47 28.42 78.5 2 +/- 0 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 3154 +/- 1160
Read 2
Lane Tiles Density (K/mm2) Clusters PF (%) Phas/Prephas (%) Reads (M) Reads PF (M) % >= Q30 Yield(G) Cycles Err Rated Aligned (%) Error Rate (%) Error Rate 35 cycle (%) Error Rate 75 cycle (%) Error Rate 100 cycle (%) Intensity Cycle 1
1 216 210 +/- 61 29.43 +/- 18.55 0.000 / 0.000 136.33 35.55 96.2 0 +/- 0 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 4668 +/- 2164
2 216 236 +/- 37 27.98 +/- 20.33 0.000 / 0.000 152.83 40.4 96.8 0 +/- 0 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 5927 +/- 1886
3 216 184 +/- 80 29.13 +/- 18.35 0.000 / 0.000 119.12 28.78 95.7 0 +/- 0 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 5167 +/- 2191
4 216 217 +/- 68 22.66 +/- 18.45 0.000 / 0.000 140.47 28.42 96.9 0 +/- 0 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00
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