Create an assembly for phased variant analysis

The purpose of phased variant analysis is to determine the specific combination of genetic variants (alleles) inherited together on a single chromosome from each parent. Phased variant analysis can be used to:

• Identify genes that are inherited together (linked) because they are located close to each other on the same chromosome. This is crucial for understanding the genetic basis of complex diseases.

• Trace the ancestry of individuals and populations through haplotype analysis.

• Determine whether or not there is at least one functional allele in cases of compound heterozygosity.

• Study the genetic diversity of populations and track the evolution of genetic variations over time.

• Predict how a patient will respond to certain medications, leading to more personalized and effective treatments.

In Lasergene, this workflow takes place in SeqMan NGen and GenVision Pro. SeqMan NGen provides a diploid phasing algorithm for analyzing long-read data sets as large as the complete human genome. Phased variants are then analyzed in GenVision Pro, which features several useful phase-optimized displays.

Create a phased assembly in SeqMan NGen:

To analyze phased variants with any type of software, you first need to create one or more assemblies using long-read sequencing data from a diploid organism. Why long-read data? Short read lacks the long-range connectivity needed for haplotype phasing and structural variations. In order to determine whether the variant occurs on one or both allele, the sequence must be long enough to span the allele. Those limitations can be overcome with long read data from Pacific Biosciences (PacBio) or Oxford Nanopore Technologies (ONT), since much of the genome can be “in phase.”

To create a phased assembly with SeqMan NGen, select the following options in the setup wizard:

1. In the Variant Analysis / Resequencing tab of the SeqMan NGen Workflow screen, choose the PacBio / Nanopore Whole genome workflow.

2. Choose any desired options on the wizard screens, with the following exceptions.
   
   
   • In the Input Sequences screen, load only long-read sequences from PacBio or Oxford Nanopore Technologies (ONT). For best results, we recommend using PacBio HiFi sequencing technology.
     
     
   • In the Analysis Options screen, check the box next to Detect SNPs and other small variants. Then choose Diploid – Phased as the Variant detection mode.
     
     

Together, these two options trigger SeqMan NGen to calculate phased variants once assembly is initiated.

3. In the Run Assembly Project screen, click either option to initiate the assembly.

Once the assembly is initiated, SeqMan NGen’s phasing algorithm identifies regions where the data has enough variability and assembles accurately so that phase “blocks” can be identified and displayed in GenVision Pro. Phase blocks are regions of the reference that could be phased into two haplotypes.

4. On the Assembly Summary screen (after assembly is complete) click Analyze and compare variants in multiple samples to open the results in GenVision Pro. There you can:
   
   
   • View phased/unphased regions across the whole genome in the Analysis and Genome views. Visualize and analyze phased aligned reads, phased blocks, phasing consistency, and phased variants using several tracks and options.
     
     
   • See a customizable table of phased variants in the Variants view.
     
     
   • Export phased consensus blocks to get the maternal and paternal haploid sequences for a gene of interest.

To learn more about downstream analysis, see the lower half of the GenVision Pro user guide topic Analyze Phased Assemblies.