03 November 2014

Illumina has recently released four lanes of NA12878 data from HiSeq X10. I was playing with this data set and found that my program had bad accuracy on two of them. I initially thought the data quality was different, so wrote some code to investigate the data quality. It turns out that my program was buggy, but the finding of the HiSeq X10 data quality might be of its own interest, which I am sharing here.

HiSeq X10 data quality

When I looked at the HiSeq X10 alignment in samtools tview, my first impression is that the error rate is visually higher than the previous Illumina data I have seen. This might be the cause of the [2-channel system][ch2] (as is opposed to the previous 4-channel). However, a closer look at the base quality suggested I might be wrong. The average base quality of these HiSeq X10 data is Q37.0 for reads mapped to chr11. This is compared favorably to NA12878 from Platinum Genomes (Q36.4) and the CHM1 data used in my paper (Q34.9).

Mean quality is in effect the the geometric mean of error rate, but what I observed in tview is the arithmetic mean. Could that be the cause? It is not. The arithmetic mean of HiSeq X10 data is Q24.4, still better than NA12878 (Q24.2) and CHM1 (Q17.6).

However, I still trust my eyes more. I started to believe the base quality HiSeq X10 reads is overestimated (or the older quality is underestimated). MAQ has a subcommand “mapcheck” to estimate the empirical base quality from read mapping. I don’t have this for BAM, so I implemented one. In this implementation, if 35% of >=Q20 bases are different from the reference base, the site is considered to be a variant site and is ignored.

My eyes are right after all. The following table shows the empirical arithmetic means of different data sets and also stratified by low (<Q20) and high base quality:

Dataset emQ % <Q20 bases emQ <Q20 emQ >=Q20
Platinum Genomes Q26 2.5% ~Q13 ~Q34
CHM1 Q25 4.3% ~Q12 ~Q33
HiSeq X10 L1 Q23 4.7% ~Q10 ~Q30
HiSeq X10 L3 Q23 4.6% ~Q10 ~Q30

We can see that the empirical quality of HiSeq X10 data is obviously lower - almost twice as low. This is consistent with my feeling. Note that on older data, the empirical quality could go higher if there were no variants. The 35% rule is not good enough.

Another a bit worrying sign of the new data is the systematic compositional bias. In particular, 68% of high-quality C bases in FASTQ should really be A but only 8% be G. There were compositional biases in older Illumina data, but not as bad. Does this affect variant calling? Crude evaluation using Genome In A Bottle (GIAB) suggests the variant calls are still decent. Nonetheless, more careful comparisons are needed to draw a definite conclusion.

Base quality resolution

Another visible difference of HiSeq X10 data is the reduced resolution of base qualities. There are only seven distinct quality values as is opposed to nearly 40 in the previous data. It has long been discussed whether this would impact the accuracy of variant calls with NCBI being one of the first advocates. I used to evaluate the effect on the 1000g low-coverage pilot and a high-coverge NA12878. As I remember (I have lost the data), 7 or even 4 quality values worked.

Reduced quality resolution has a positive effect on the size of alignment. Typically, for 35X human data, the size of the final BAM file is about 100GB. The size of these 35X HiSeq X10 data is only 70GB. A 30% reduction. Could we push further, say 1-bit quality?

I did an experiment. In the aligned BAM, I turned all quality below Q20 to Q10 all quality no less than Q20 to Q30 (based on the table above). I run GATK-HC on the original BAM and the quality-reduced BAM. If I compared the exact variant coordinates, HC called 7,253 unfiltered variants only present in the original BAM and 15,435 variants only in the quality-reduced BAM. The difference is minor if we notice that the difference between lane 1 and 7 is over 110,000. On GIAB, the two call sets are largely indistinguishable. In all, 1-bit quality does not obviously reduce the accuracy of variant calls.

One-bit quality further reduces the BAM size down to 40GB. In the CRAM format, it is 16GB. This is a significant reduction from typical 100GB BAMs at 35X coverage.

Although I have not tried, I firmly believe that we cannot discard base quality at all. HiSeq produces recurrent errors. These errors are usually correctly assigned to low base quality. If a variant caller ignores base quality, it is likely to make calls at these systematic errors. One bit is the minimum.

While I was doing my small experiment, I learned from the GA4GH mailing list that Illumina is also exploring the possibility of 1-bit quality. I believe this strategy should be fine for normal samples. I actually think, cautiously, that 1-bit quality may even work for cancer data, but I am not experienced enough to confirm this.

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