Read Preprocessing Algorithms¶
In ADAM, we have implemented the three most-commonly used pre-processing stages from the GATK pipeline (DePristo et al. 2011). In this section, we describe the stages that we have implemented, and the techniques we have used to improve performance and accuracy when running on a distributed system. These pre-processing stages include:
- Duplicate Removal: During the process of preparing DNA for sequencing, reads are duplicated by errors during the sample preparation and polymerase chain reaction stages. Detection of duplicate reads requires matching all reads by their position and orientation after read alignment. Reads with identical position and orientation are assumed to be duplicates. When a group of duplicate reads is found, each read is scored, and all but the highest quality read are marked as duplicates. We have validated our duplicate removal code against Picard (The Broad Institute of Harvard and MIT 2014), which is used by the GATK for Marking Duplicates. Our implementation is fully concordant with the Picard/GATK duplicate removal engine, except we are able to perform duplicate marking for chimeric read pairs.  Specifically, because Picard’s traversal engine is restricted to processing linearly sorted alignments, Picard mishandles these alignments. Since our engine is not constrained by the underlying layout of data on disk, we are able to properly handle chimeric read pairs.
- Local Realignment: In local realignment, we correct areas where variant alleles cause reads to be locally misaligned from the reference genome.  In this algorithm, we first identify regions as targets for realignment. In the GATK, this identification is done by traversing sorted read alignments. In our implementation, we fold over partitions where we generate targets, and then we merge the tree of targets. This process allows us to eliminate the data shuffle needed to achieve the sorted ordering. As part of this fold, we must compute the convex hull of overlapping regions in parallel. We discuss this in more detail later in this section. After we have generated the targets, we associate reads to the overlapping target, if one exists. After associating reads to realignment targets, we run a heuristic realignment algorithm that works by minimizing the quality-score weighted number of bases that mismatch against the reference.
- Base Quality Score Recalibration (BQSR): During the sequencing process, systemic errors occur that lead to the incorrect assignment of base quality scores. In this step, we label each base that we have sequenced with an error covariate. For each covariate, we count the total number of bases that we saw, as well as the total number of bases within the covariate that do not match the reference genome. From this data, we apply a correction by estimating the error probability for each set of covariates under a beta-binomial model with uniform prior. We have validated the concordance of our BQSR implementation against the GATK. Across both tools, only 5000 of the 180B bases (\(<0.0001\%\)) in the high-coverage NA12878 genome dataset differ. After investigating this discrepancy, we have determined that this is due to an error in the GATK, where paired-end reads are mishandled if the two reads in the pair overlap.
- ShuffleRegionJoin Load Balancing: Because of the non-uniform distribution of regions in mapped reads, joining two genomic datasets can be difficult or impossible when neither dataset fits completely on a single node. To reduce the impact of data skew on the runtime of joins, we implemented a load balancing engine in ADAM’s ShuffleRegionJoin core. This load balancing is a preprocessing step to the ShuffleRegionJoin and improves performance by 10–100x. The first phase of the load balancer is to sort and repartition the left dataset evenly across all partitions, regardless of the mapped region. This offers significantly better distribution of the data than the standard binning approach. After rebalancing the data, we copartition the right dataset with the left based on the region bounds of each partition. Once the data has been copartitioned, it is sorted locally and the join is performed.
In the rest of this section, we discuss the high level implementations of these algorithms.
|In a chimeric read pair, the two reads in the read pairs align to different chromosomes; see Li et al (Li and Durbin 2010).
|This is typically caused by the presence of insertion/deletion (INDEL) variants; see DePristo et al (DePristo et al. 2011).