6 Month Allelic Series RNAseq QC

6 Month Allelic Series RNAseq QC

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QC summary

QC was performed on all 192 samples focusing on determining failed or outliersamples. Four samples are recommended for omission from the final analysisdataset based on evidence of RNA degradation, PCA analysis, and model-basedgene outlier detection. Those four samples can be found on slide 19.

Additionally two correctable issues were identified. First, one flowcell worth of samples was run an additional time to add read depth to the 100 million required.This re-run was inadvertently run as 75-mers instead of 50-mer so the samplesare a mix of read length. Secondly, for a subset of cortex samples (Q92 and Q140)there appears to be an infinitesimal but detectable amount of liver tissue. Theoverall dilution is 500-1000x, but given the extraordinary sensitivity of RNAseq thisis still measureable. We have recommended a simple filter to remove those liver transcripts based on the fact that they have a recognizable correlation pattern (listed on slide 29), but other methods may be more sensitive.

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How does CHDI QC RNAseq data in general?

• Mostly we’re looking for outliers• Also showing overall experiment

worked• When we find outliers, we try to

determine the cause– That helps show it is an outlier and

not part of the biology• Methods

– Principal Components Analysis– RNA degradation plots– Paired end insert size– Read lengths– Read mapping efficiency– Repetitive sequences and their origin– Highly expressed genes– # gene outliers

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PCA whole datasetColor by Tissue

cortexLiverstriatum

Shape by SexFM

• Not surprisingly, tissuescluster

• Strong sex effect in liver• Cortex is tightly clustered

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PCA striatumColor by Q Length

Q111Q140Q175Q20Q80Q92

Shape by GenotypeHET

• Q lengths cluster, good sign the design worked

• Q92, 111, 140, 175 uniquelycluster

• They even stagger in Q length order

• Couple potential outliers (in red outline)

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PCA cortexColor by Q Length

Q111Q140Q175Q20Q80Q92

• Only Q175 stands outsidethe main cluster

• Possible Q175 outliers,but hard to be certain

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PCA liverColor by Q Length

Q111Q140Q175Q20Q80Q92

Shape by SexFM

• Strong sex clustering willneed to be accounted for

• No strong Q clusters (sex masking?)

• One potential outlier

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Duplication in brain (representative examples)

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• Duplication is consistent andhovers between 13-24%

• No red flags• Higher in striatum than

cortex generally• Origin of the majority of the

duplicated sequences is mitochondrial

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Liver duplication (representative examples)520_Liver_Q111_HET_M_L8.LB12_1.clipped.fastq


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• Liver duplication is much higher, 40-50%

• Major duplicated sequences are all mouse pheromone receptors (Mup1-21)

• Hurts our true read depth, but nothing terrible

• Should keep in mind for future liver work

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20914_449L_striatum_Q175_WT_M_L1.LB1_1.clipped

Bin1 Bin18 Bin35 Bin52 Bin69 Bin860

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5’ -> 3’ degradation charts (representative examples)Color by TranscriptBin

1-499500-9991000-19992000-29993000-39994000-49995000+

Displays the likelihood of gettingfull length transcripts for variousmRNA lengths• Very high quality samples in general• Most samples show >70% of all mRNA

molecules are >80% complete• Liver on average more degraded• Some samples have degradation in the

longer mRNA species (one marked in red)

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Suspect samples by 5’ -> 3’ degradation

454_Liver_Q175_HET_M_L3._1.clipped456_Liver_Q175_HET_M_L7.LB4_1.clipped20947_452L_cortex_Q175_HET_M_L8.LB2_1.clipped845_Liver_Q92_HET_F_L6.LB25_1.clipped522_Liver_Q111_HET_F_L8.LB13_1.clipped452_Liver_Q175_HET_M_L1.LB2_1.clipped776_Liver_Q140_HET_F_L8.LB13_1.clipped716_Liver_Q80_HET_F_L7.LB6_1.clipped843_Liver_Q92_HET_F_L6.LB23_1.clipped21051_460L_cortex_Q175_HET_F_L3.LB6_1.clipped

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GC content per read has a red flag20914_1_449L_striatum_Q175_WT_M_L1.LB1_1.clipped.fastq

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8 of the samples have a “shoulder”in the GC# chartThis is usually a really bad thing• Suggests non-mouse or

non-biological sequence

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Those same samples flag for read length as well20914_1_449L_striatum_Q175_WT_M_L1.LB1_1.clipped.fastq

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Those same samples have a mixof 50mer reads and 75mer readsThat’s very odd

At this point we asked our sequencinglab for clarification on what happened

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Our sequencing partner found the cause

264401 20921_1_450L_cortex_Q175_HET_M 20130523 V02604 VIRT 2264416 23535_1_528L_cortex_Q111_HET_F 20130523 V02604 VIRT 3264418 28243_1_644L_cortex_Q20_HET_M 20130523 V02604 VIRT 4264397 35624_1_844L_striatum_Q92_WT_F 20130523 V02604 VIRT 1264447 35631_1_845L_cortex_Q92_HET_F 20130604 V02761 VIRT 1264448 35657_1_847L_cortex_Q92_HET_F 20130604 V02761 VIRT 2264451 454_Liver_Q175_HET_M 20130604 V02761 VIRT 3264455 462_Liver_Q175_HET_F 20130604 V02761 VIRT 4

The 8 suspect samples

For these 8 samples, the initial run didn’t get a full 100 million reads. When that happens the lab runs the samples again and then merges the run into a full “virtual run” of the full read depth we paid for. That’s all good. The strange thing that happened to us this time was that the run they added our 8 samples to (they add it to ongoing flow cells) happened to be a 75mer run. Again no big problem usually, and what they do is clip off 25 bases in their processing and all is compatible. This specific time they forgot to trim, so we saw the ugly intermediate state. What this means is that the data for those 8 are fine. They are longer, but still good reads from our samples.

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Mitochondrial rate in brain

8-9% of the reads are mtRNAnothing surprising there

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Mitochondrial rate in liver

6-7% of reads are mtRNAAgain in line with expectations

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Other QC parameters that looked great

• Insert sizes: All right around 175 as expected• Sense/antisense sequence ratio: 1:1 as expected• Sequence coverage

– 40% of mouse transcriptome detected in brain– About 30% of mouse transcriptome detected in liver

• Mapped read rate in the upper 90s – 98% for brain, 96% for liver

• 95-97% of our reads are mapped to known genes– 3-5% intergenic regions

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Model based outlier detection

20914_449L_striatum_Q175_WT_M_L1.LB1_... 23385_516L_striatum_Q111_HET_M_L1.LB2... 28295_648L_cortex_Q20_HET_M_L8.LB21_1... 30772_715L_striatum_Q80_WT_F_L3.LB11_... 33205_781L_striatum_Q140_WT_F_L6.LB6_...0

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Method by which we look for the number of genes that areoutliers after accounting for our modeled effects• 2 samples stand out, and additional 4-6 are suspect, but probably

OK (Q92 Het males)



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Integrating the sample QC to choose omissions

A very simple way to determine what to throw out is to look for multiple strikes against a sample

454_Liver_Q175_HET_M_L3._1.clipped456_Liver_Q175_HET_M_L7.LB4_1.clipped20947_452L_cortex_Q175_HET_M_L8.LB2_1.clipped845_Liver_Q92_HET_F_L6.LB25_1.clipped522_Liver_Q111_HET_F_L8.LB13_1.clipped452_Liver_Q175_HET_M_L1.LB2_1.clipped776_Liver_Q140_HET_F_L8.LB13_1.clipped716_Liver_Q80_HET_F_L7.LB6_1.clipped843_Liver_Q92_HET_F_L6.LB23_1.clipped21051_460L_cortex_Q175_HET_F_L3.LB6_1.clipped

5’ -> 3’ charts30811_718L_striatum_Q80_HET_F_L5.LB14_1.clipped




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Model based outliers

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Final list of proposed samples for omission

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Liver contamination in cortex Q140 and Q92?

While the sequencing lab was looking into the 75mer issue I ran cortex through some basic statistical modeling (omitting the samples mentioned previously)

I found changes, but the pattern and biology was all wrong

Alb

ENSMUSG000000293680

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Color by Q LengthQ111Q140Q175Q20Q80Q92

Every single change is an increase

Completely off in Q175, 111, 80, and 20On (but not that strongly in 140 and 92)

It’s make no sense for Q111 to be skippedand for Q175 to go back to normal

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Albumin is the top hit?Isn’t Albumin liver specific?

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Some of the other changed genes are suspicious

• Albumin• ApoC3, C1, • Mup3, 10, 18, 19• FABP1• Urate oxidase

All reasonably solid liver markers

DAVID functional annotation also suggests the altered genesare liver related (p < 10-5)

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A subset of genes with good correlation between liver and cortexbut shifted from the 1:1 axis

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Same chart with the “significant” genes in red

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Same chart and shading in Q111, notice the Lack of linear correlation

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What we suspect happened

• The basic problem is that liver specific transcripts should not have correlated expression in cortex

• A very small amount of liver contamination has occurred. The shift is 500 to 1000 times lower than normal liver expression

• What this means is only the absolute highest liver expressed genes are detected at all

• The challenge is uniquely identifying the affected genes

Cortex Striatum Liver

Albumin 173 0.01 40979

FPKMs of albumin, which should not exist in brain

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Liver filter created as• Liver mean count > 2000• Mean ratio of liver to cortex > 500• Cortex count > 0

Not a bad first approximation

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Effect of filtering out the liver specific genes from the cortex data

Q80 Q92 Q111 Q140 Q1750

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Hits pre-filterHits post-filter

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Summary of QC

• All but 4 of the 192 samples can move forward to the analysis

• A filter to clear out highly expressed liver genes is needed for the cortex Q140 and Q92 sets

• Striatum PCA plots show that CAG length is the single largest global element of variance!

6 Month Allelic Series RNAseq QC

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