Dinh/Dinh 2013/NOTES/2013-10-7
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Blueprint Capture, low mapping issue
- Low mapping can be caused by any of the following problems
- Contamination - contamination of library after capture would not be amplified by the last PCR step or sequenced due to lack of adapter sequences. Contamination of reagents during capture may lead to low mapping rates. Contamination may explain why we have such variable mapping rates, since each DNA sample had varying quality, it's possible that the lower quality samples would have more capture from contaminating sequences and thus lower mapping rate.
- Do the amount of input converted gDNA correlate with mapping rate?
- Do the captured sequences map to other genomes in NT database?
- Chimeric capture sequences - When imperfect hybridization of capture arms to genomic sequences which occurs frequently in the genome, leading to off-target & chimeric sequences. Or when the capture arms capture each other.
- How many of the sequences map to the adapters, linkers, and primers?
- Do the sequences map to human repeats? - No, when mapping with Bowtie, most of the reads either map uniquely or not at all.
- Do the sequences map to non-human repeats? - Check by performing Blast to RepBase18.08
- Error from sequencing - We did not use a PhiX control for the MiSeq runs, but had a control for the HiSeq RapidRun (control was ~ 89% of flowcell, BP libraries was ~ 11%). The mapping rate of the HiSeq RapidRun was still low for many samples. Also, the majority of mapped reads have low error rates at the 3' end.
- Error from oligo synthesis - error from oligo synthesis would still allow us to recover the alignments from the off-target regions. Even so, we should get lower the specificity of capture, but this is not the case.
Reads sampling
- Randomly sample 1000 reads from the following libraries for analysis:
1. Blueprint samples #1 and #2 from the Aug 28 MiSeq run. (compare different BP samples) 2. Technical replicates for Blueprint samples #1 and #2 from the Aug 28 MiSeq run. (compare different captures) 3. Technical replicates for Blueprint samples #1 and #2 from the Sep 13 HiSeq Rapid run. (compare different runs) 4. NA12878 captured with the original probes set and sequenced in HL157 (+ control) 5. NA12878 captured with the normalized probes set and sequenced in HL157 (+ control) 6. DMR330K capture from the Mar22 MiSeq run as positive control. (+ control)
- All reads were sampled from the first read from each run.
BLAST sequences to adapters, linkers, and primers
- Since these were not bisulfite converted, I use the original reads to blast.
- Library free adapters/linkers/primers
>AmpF6.3Sol AATGATACGGCGACCACCGACACTCTCAGATGTTATCGAGGTCCGAC >AmpR6.3Ind1 CAAGCAGAAGACGGCATACGAGATCGTGATGCTAGGAACGATGAGCCTCCAAC >SolSeqV6.3.2 ACCGACACTCTCAGATGTTATCGAGGTCCGAC >SolSeqV6.3.2r GCTAGGAACGATGAGCCTCCAAC >AmpR6.3IndSeq GTTGGAGGCTCATCGTTCCTAGC >AmpF6.4Sol AATGATACGGCGACCACCGAGATCTACACCACTCTCAGATGTTATCGAGGTCCGAC >SolSeqV6.3.3 TACACCACTCTCAGATGTTATCGAGGTCCGAC
BLAST sequences to NT database
- If capture reagents were contaminated, we would expect that reads from capture of the converted sample would not map to the unconverted NT database. Reads that map to the unconverted NT database would means that we have contaminated capture reagents.
BLAST sequences to RepBase
- If there were off-target capture to repeats, we would get hits to the converted RepBase sequences, but they would be mostly human.
- Reads were encoded by converting G to A (all read 1 would be on the complement strand)
- BLAST args:
$blastn -p blastn -d $db_dir/all_repeats.bis.fwd -K 100 -e 0.0001 -g F -F "m D" -a 4 -W 7 -i $f.encoded_2 -o $f.bis.fwd.all_repeats.blast