**1.3. NGS HLA typing**

#### *1.3.1. Pros and cons of switching to NGS HLA typing*

One of the advantages of switching to NGS HLA typing is that inherent phasing ambiguities present in Sanger sequencing can be eliminated. As mentioned before, the two chromosomes produce separate reads, and an adequate bioinformatics workflow can separate these reads and assemble them into phased consensuses. Furthermore, using modern kits, it is not only possible to sequence the most polymorphic exons, but whole genes and many loci can be typed at once. This whole-gene sequencing approach provides an unprecedented precision, reveal‐ ing novelties mainly in intronic and untranslated (UTR) regions. On the other hand, the high amount of data, the fundamentally different NGS workflow needs not only new laboratory equipments and reagents, but some bioinformatics and IT skills: sequence search, alignment, read filtering, database handling, etc. are among the daily routines of a HLA lab practitioner. The amount of generated data is more by magnitudes compared to the size of Sanger traces, and validating novelties by confirmatory typing can be cumbersome. In a low-throughput laboratory processing the samples in the wet lab have to be planned in advance; many kits accommodate more samples than the amount accumulating during a week/month.


**Table 1.** Main advantages and disadvantages of NGS HLA typing.

#### *1.3.2. NGS HLA typing methods*

Algorithms and kits for genotyping the HLA loci using NGS reads are in the focus of several publications in recent years [9]. Some of the authors use the straightforward read alignment followed by the variant call approach [10], and others developed designated genotyping algorithms for a wide variety of kits and sequencing approaches [11–14]. Since some of these authors are more interested in primer and sequencing workflow development, and others address the genotyping/bioinformatics problems concerning HLA typing, there is already a high diversity of available workflows.

The pioneering publications for NGS HLA typing were already considering targeted longrange PCR amplification and quality check measures [15–17] such as strand bias, though some cases managed to achieve high concordance for two fields only by using population frequency information. The ultimate goal is to have a primer set and a wet-lab and bioinformatics workflow to get phased, whole-gene consensus sequences with unambiguous four-fields typing [18, 19].

**1.3. NGS HLA typing**

*1.3.1. Pros and cons of switching to NGS HLA typing*

372 Next Generation Sequencing - Advances, Applications and Challenges

**Pros Cons**

**Table 1.** Main advantages and disadvantages of NGS HLA typing.

Unprecedented precision: We do know that HLA expression is heavily affected by introns/UTRs, we are getting an insight into these sequences as well

*1.3.2. NGS HLA typing methods*

high diversity of available workflows.

One of the advantages of switching to NGS HLA typing is that inherent phasing ambiguities present in Sanger sequencing can be eliminated. As mentioned before, the two chromosomes produce separate reads, and an adequate bioinformatics workflow can separate these reads and assemble them into phased consensuses. Furthermore, using modern kits, it is not only possible to sequence the most polymorphic exons, but whole genes and many loci can be typed at once. This whole-gene sequencing approach provides an unprecedented precision, reveal‐ ing novelties mainly in intronic and untranslated (UTR) regions. On the other hand, the high amount of data, the fundamentally different NGS workflow needs not only new laboratory equipments and reagents, but some bioinformatics and IT skills: sequence search, alignment, read filtering, database handling, etc. are among the daily routines of a HLA lab practitioner. The amount of generated data is more by magnitudes compared to the size of Sanger traces, and validating novelties by confirmatory typing can be cumbersome. In a low-throughput laboratory processing the samples in the wet lab have to be planned in advance; many kits

accommodate more samples than the amount accumulating during a week/month.

Multiple loci sequenced in one sample Loads of data, needs serious IT infrastructure

High-throughput lab workflow, more samples to process In a low-throughput lab have to plane forward

Phasing problem inherent in Sanger traces is not present There are still remaining ambiguities; some bioinformatics

Algorithms and kits for genotyping the HLA loci using NGS reads are in the focus of several publications in recent years [9]. Some of the authors use the straightforward read alignment followed by the variant call approach [10], and others developed designated genotyping algorithms for a wide variety of kits and sequencing approaches [11–14]. Since some of these authors are more interested in primer and sequencing workflow development, and others address the genotyping/bioinformatics problems concerning HLA typing, there is already a

The pioneering publications for NGS HLA typing were already considering targeted longrange PCR amplification and quality check measures [15–17] such as strand bias, though some cases managed to achieve high concordance for two fields only by using population frequency information. The ultimate goal is to have a primer set and a wet-lab and bioinformatics

skills are desired

Many novelties, mainly in introns

Other approaches are trying to extract HLA types from existing whole-exome (WES), wholegenome (WGS), or even RNA-Seq data. A short review of diverse methods addressing WES and WGS reads can be found in [20], exploring how to tackle problems regarding HLA gene homology (cross-mapping reads, see below) and missing intronic information.

It is expected that the number of both the kits and the typing algorithms will grow in the near future, and laboratories will use more than one strategy for confirmatory testing (for a comprehensive list of available HLA typing software see Table 2). Therefore, our goal was to give details about the possible source of ambiguity and mistyping.


**Table 2.** Collection of available HLA typing software for NGS data. Entries with a star (\*) are considered obsolete, their web pages have not been updated for more than two years.
