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Athurva Gore/LabNotes/2009-8-24
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{{ AGLabEntry|2009-8-21|2009-8-25 }} =Probe Sets= * '''FLYDUP5279''' has finished; all old probesets are done * Need to create and send invoice to Zymo today ** Also mention that probe generation can be rerun without quality filters? * Need to redo TDMR set and Cpg-SNP set, but with a new probe design... ** Constrain arm+target size to 105 bp ** Then modify P2MIP_v9 to use a 45 bp total arm size ** This will allow library construction to not be necessary *** During capture, MME1 digestion removes 17-18 bp from each side of H1_TARGET_H2 *** Should ideally leave us with 75 bp segment remaining, 60 bp of which will be target * This can be read in the sequencer without library construction, allowing for a much quicker and more efficient experimental procedure =Exome Read Analysis= * Turns out that HL_003 had very poor focus quality. Cannot trust the data because of this, as calls may have been mislocated. * Flowcell was reused for HL_006; same capturing experiment, but much higher quality data (microscope was in focus) * Analyzing reads from HL_006 ** Comparing Lane 4 and Lane 5 ** '''Lane 4 - NA12878, size-selected''' ** '''Lane 5 - NA12878, no size selection''' * Mapped using Bowtie, called SNPs using SAM * Compared to 1KG and dbSNP * '''Results BEFORE ANY FILTERING:''' ** '''Lane 4''' - Size Selection *** vs 1KG, 4,044 hits, 1,533 misses, 218 miscalls (right location but wrong call) *** vs dbSNP, 4,309 SNPs in dbSNP, 1,492 not in dbSNP ** '''Lane 5''' - No Size Selection *** vs 1KG, 3,935 hits, 21,493 misses, 305 miscalls (right location but wrong call) *** vs dbSNP, 4,411 SNPs in dbSNP, 21,328 not in dbSNP * Wanted to investigate why this was happening. * Could junk be getting appended to reads, causing successful mapping to produce errors? ** HERE, TESTED ONLY AGAINST DBSNP. ** Hypothesis: Extra bases are being ligated onto the start or end of captured regions, causing mapping to be successful but produce many homozygous SNPs ** Checked both s4 (size selected) and s5 (not size selected) ** '''Results, no filtering''' *** S4: Misses have on average 28% start/end reads, Hits have on average 7% start/end reads *** S5: Misses have on average 42% start/end reads, Hits have on average 8% start/end reads ** Bases being added to beginning or end of reads could be causing issues? ** Checked the reads themselves. ** Saw a large number of reads with an error in the "39th" position (ie either the start or end of mapped read depending on orientation) *** S5 (not size-selected) had 153286, while S4 (size-selected) had 50140 *** Size-selected version had much fewer of these errors. ** Could it just be 3' sequencing problems? *** Would not make as much sense, as multiple reads had the exact same error for many of the erroneous SNP calls. *** Would get results like 30-40% reads are at start/end position? Could be artifacts from low-coverage SNPs. ** Checked with a coverage filter of > 4: *** S4: Misses have on average 12% start/end reads, Hits have on average 7% start/end reads *** S5: Misses have on average 29% start/end reads, Hits have on average 6% start/end reads ** This is a bit harder to read. It appears that problem is no longer existent in size-selected case, but with no size-selection, problem still exists. * '''CHECK WHILE REMOVING CLONAL READS''' ** This statistic is most likely due to clonal reads still being counted under Dr. Zhang's script. ** Tried re-running on s4 and s5 using SAMtools' rmdup to remove clonal reads ** Check if this works. * rmdup does not seem to be working ** Modified downSampler code to only keep one of each read type ** Then check calls; see if we do better ** Might do a lot better, as many errors seem to arise from ends of reads... * No longer have multiple reads starting and ending in the same position. ** Should hopefully perform better ** Now comparing to 1KG to check
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