Kun:LabNotes/MONOD/2014-3-18
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MONOD Round 3[edit]
Target identification[edit]
- The specificity of the Round 2 probe set seemed to be very low. Dinh found that 42.5% of the captured targets overlap with repeats. This could explain many off-target reads.
- In this re-design, I included the RRBS data (ENCODE, GSE52140) for the A549 and HTB56 lung cancer cell line for identifying cancer DMS. In addition, I also added ENCODE RRBS data from normal brain, lung and pancreas as the controls.
./find_DMS_PANC_GBM_LC_v3.pl > GBM_PC_LC_DMS_v3.txt ../extract_clusters.pl GBM_PC_LC_DMS_v3.txt > GBM_PC_LC_DMS_clusters.v3.txt
Probe design[edit]
- I pretty much followed the same design as the MONOD Round 2 probes.
- Input files: GBM_PC_LC_DMS_clusters_v3_input_plus.txt;GBM_PC_LC_DMS_clusters_v3_input_minus.txt
- Job files:jobFile_GBM_PC_LC_DMR_150_plus.pl;jobFile_GBM_PC_LC_DMR_150_minus.pl
- ppDesigner output: GBM_PC_LC_DMS_probes_v3.txt
- Removal of probes capturing less than 4 CpG sites
../filter_probe.pl < GBM_PC_LC_DMS_probes_v3.txt > GBM_PC_LC_DMS_probes_v3_4cpg.txt
- Dinh helped me to annotate the repeat content of these probes.
- In the final 12k oligo pool, I included all 6466 non-repetitive probes in this set, plus additional 5534 non-repetitive fetal DMR v2 probes in chrs 13/18/21/X to fill up the space.
- ProbeInfo file:MONOD_V3_12k_probeInfo.txt
- Oligo sequenced ordered (V6 primers, N8 UMI):MONOD_V3_12k_seq_only.txt