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Routine analysis

Routine analysis can be summarised in order as raw sequence data quality control, read trimming, reference alignment, subsequently followed by the GATK best practices for SNV and indels. Figure “analysis flow” illustrates the basic analysis workflow structure. Proceeding top to bottom, the procedure making up the left side of fig. [fig:analysis_flow] contains the procedures for routine analysis. Each rectangle box labels a program function, key input and output data are shown with light slanted boxes. The most important data retention steps are indicated with a “data store” symbol. The right-hand side of the same figure illustrates the second phase of analysis used in this study; tailored analysis, or cohort-specific analysis. The annotation, filtering, and segregation of data here depends on the project. A generally useful strategy will output gene candidate data based on inheritance type to produce individual datasets for each (i) functional heterozygous variants (including de novo, somatic, known dominant genes, etc.), (ii) homozygous only variants, and (iii) potential compound heterozygous variants, and (iv) a master version of all variants that have completed the filtering pipeline. These datasets are generally small (<1MB per individual) and combined into all individuals per sequence run or cohort.

Genome and exome analysis is an iterative process. Although there are routine steps, different methods will be used depending on each experiment. Data storage is a major factor in genetic analysis. Not only are the initial files large in size, many intermediate files are produced and may themselves be important to retain for a certain period. Key output files are shown by light slanted boxes. As shown in Figure [fig:analysis_flow_storage], storage structure is divided between long-term and short-term storage. A /work/ directory is used for long-term storage and is backed up routinely. Short-term storage is used for intermediate files which are held in /scratch/ directories and not backed up. File sizes are represented by colour, dark orange indicating large and light yellow indicating small sizes.

Analysis workflow structure.

Tools used are shown in square boxes. Reference data used secondary to inputs are shown as light boxes with curved sides. Key output files are shown by light slanted boxes. Storage structure is divided between long-term and short-term storage. The same figure key is applied to Fig. [fig:analysis_flow_storage].

FigureLabel analysis_flow


Analysis storage structure

Storage structure is divided between long-term and short-term storage. A /work/ directory is used for long-term storage and is backed up routinely. Short-term storage is used for intermediate files which are held in /scratch/ directories and not backed up. File sizes are represented by colour, dark orange indicating large and light yellow indicating small sizes. Figure key is shown in Fig. [fig:analysis_flow]. FigureLabel analysis_flow_storage

Sequence alignment to reference genome

The analysis methods are normally run as a pipeline workflow. The basic methods do not have major changes in theory, although there are usually several methods or software options available for each step. Once a working pipeline is established, most of a researcher’s time can be spent on the tailored analysis at the end of the pipeline, which requires more specialised steps. Each individuals’ exome sequence data contains approximately 3-8 GB of raw data. This is output as  150bp raw unmapped sequence fragments that must be aligned to the reference human genome. The raw sequence data is normally collected into a fasta format file called a “fastq” file (pronounced “fast” “q”).

An important consideration for sequence analysis is the reference genome used for comparison. The coordinates for individual nucleotides vary between reference versions. For example, aligning with one reference version will produce a file that contains chromosome, position, and variants specific to that genome reference. Annotation will be required to interpret results, but if databases based on coordinates from different reference versions are used during this step the results will be incorrect.

The current human genome reference is a version of Genome Reference Consortium Human Build 38 patch release 13 (GrCh38) ( Because of the timing when next generation sequencing became popular, many researchers tend to use genome build GrCh37 in their analysis
( However, it is preferable to use the more recent GrCh38. A lot of the best standardised methods that are used in the field were developed while genome build GrCh37 was the most recent version. Thousands of database samples will be in storage which have been aligned with this reference. Bioinformatic analysis is extremely more powerful when comparing many samples than when looking at one sample individually. Therefore, many people still tend to align their data to GrCh37 so that they can use their reference databases without going back and realigning all of their old samples again to GrCh38. The most popular method for aligning short read data to the reference human genome is “BWA-MEM” (a Burrows-Wheeler transformation aligner) (missing reference)” width=”40%”>

Read deduplication

Sequence library preparation may contain a PCR amplification step. Individual fragments of genomic DNA will be amplified. If a read contains a variant then, after amplification, we only want to count this occurrence once so that we do not interpret an inflated allele depth. Therefore, identical reads are marked as duplicates. Alternative overlapping reads that also contain the same variant will result in detection of a true germline variant. When no other overlapping reads contain the variant then the allele depth will remain low and be filtered out later by a frequency threshold, or flagged as potentially somatic. [subsec:text_dedup] For command line usage examples of this step, see page .

Read realignment and targets

After sequence alignment, regions of misalignments will inevitably exist. To deal with this feature, a local realignment process is used such that the number of mismatching bases is minimized across all the reads. This main source of misalignments corrected in this step are due insertions and deletions. Current versions of the GATK suite no longer require this step as it is integrated into the downstream process of haplotype assembly (via HaplotypeCaller or MuTect2). However, the step is included here since it is a well known legacy feature and is a very useful concept to understand for new users. As usage example is provided on page .

Base quality score recalibration

The alignment steps are difficult and computationally intensive. There are methods to double check the alignment and see if more appropriate corrections can be made. Once the quality control is all done, we are left with a Bam file format which is ready for variant analysis. Most of the bioinformatic community agrees on some best practices using the tools maintained by the Broad Institute. The GATK is widely used for the QC and variant analysis of genomic data.

Joint analysis of multiple samples increases the accuracy of our methods. Not only are the algorithms checking for consistencies in the data, but sometimes the sequence library preparation induces errors in the sequences produced. For example, sometimes a particular nucleotide position can be sequenced incorrectly. In isolation we would expect that this patient has a true mutation in the gene, but when we compare the whole cohort we see that it is just a common sequencing artefact.

When we look at the number of variants compared to the reference genome there can be hundreds of thousands. The vast majority of these can be ignored by [1] comparing the in-house database of false positive, [2] comparing the unrelated samples sequenced on the same run to remove library preparation errors, [3] compare to databases of common polymorphisms.

In genome wide association studies, researchers are generally looking at the mild effects of common polymorphisms which occur in the general population and may associate with a particular phenotype. In rare disease analysis we are focusing on the very rare variants that have a strong effect to produce a severe phenotype. Therefore, another step for pruning out the data is to compare to large cohorts of “healthy” populations to leave only the very rare variants in our dataset. The command line arguments can be see on page .

Haplotype calling

The final output, illustrated in the GATK best practices figure above, is stored in a Genomic Variant Call Format (GVCF). The GVCF file type that now presents our data has one row for each nucleotide along the genome. The row contains the DNA position, the nucleotide (either wild-type (ref) or mutation (alt)) and lots of quality and metrics information. We analyse variants against curated databases of known mutations. We also analyse again separately for indels, since a shift in the sequence position due to a indel could affect the alignment accuracy. For an example see page .

Cohort joint genotyping

We can merge 10-100s of samples together by combining the files to simplify how we handle the data. Tracking hundreds of files is exponentially more difficult than tracking 1. The GVCF contains a row of data for every single nucleotide. We can condense the information by converting to a VCF which instead only keeps information for every variant but not every wild type nucleotide (since wild type is healthy and of no interest to us). The GATK documentation provides a great explanation of the shared features and differences between gVCF and VCF files.

As our dataset becomes smaller we can double check to focus on just the most likely disease-causing mutations. Often times, a research group or clinical research team will collect genetic material from patients who they would like to diagnose genetically, or even collect a great database of patients with a shared phenotype. There are many of facilities that will sequence the samples commercially. When one orders exome or whole genome sequencing commercially, most facilities will also provide data analysis.

The output of their analysis is usually this VCF file (mostly contain the chromosome, nucleotide position, and a selection of quality control information). This file is usually the end-point of routine analysis. However, it does not really put one in a position for a genetic diagnosis. Very good services will also provide lists of top candidate genetic determinants along with information on each of the genes and possible mechanisms of pathogenicity (although the number of companies doing high-level tailored analysis is small but growing). There are usually more hurdles in determining candidate variants of unknown significance. An example of the command line arguments used can be found on page .