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Trusight HLA

Questions & Answers

 

General

What is the expected level of resolution?

This sequencing panel provides high-resolution HLA typing results to at least 2 fields of resolution and usually achieves 3 or 4 fields of resolution depending on the allele and the HLA nomenclature available.  

Are there known ambiguities resulting from TruSight HLA v2 sequencing?

There are 3 amplicon ambiguities found using the IMGT/HLA 3.23 database with TruSight HLA v2.
DRB1*12:01:01 and DRB1*12:10 are ambiguous due to the forward primer location. DRB1*12:01:01 and DRB1*12:10 are both part of the DRB1*12:01:01 G group. They are distinguished from each other in a single base position in Exon 1 (IMGT Codon -16, exon 1 base position 40). The DRB1*12:10 codon -16 is ATT (Ile) and the DRB1*12:01 codon is GTT (Val). The TruSight HLA v2 DRB1 amplicon begins in intron 1 and does not cover exon 1. Coverage of exon 1 was excluded from the assay because it would require a 14 kb amplicon that would be challenging to amplify reliably.
DPB1*13:01:01 and DPB1*107:01 are ambiguous for similar reasons to the ambiguity previously described. DPB1*13:01:01 and DPB1*107:01 are both part of the DPB1*13:01:01 G group. The TruSight HLA v2 amplicon does not cover exon 1. These alleles are distinguished from each other at 2 base positions in exon 1(IMGT Codon -22, base position 24 and Codon -14, base position 47). Codon -22 is GCG (Ala) in DPB1*13:01:01 and GCA (Ala) in DPB1*107:01. Codon -14 is ACG (Thr) in DPB1*13:01:01 and ATG (Met) in DPB1*107:01. Coverage of exon 1 was excluded from the assay because it would require an amplicon that would be challenging to amplify reliable.
DRB1*08:01:01 and DRB1*08:01:03 are ambiguous in IMGT/HLA database version 3.23. As of March 2016 and the release of IMGT/HLA 3.24, 08:01:03 allele has been removed from the database because “sequence shown to contain errors and be identical to DRB1*08:01:01.”
In addition to these amplicon ambiguities, conditional ambiguities arise in DPB1 and DQB1. We define a conditional ambiguity as an ambiguity present only when 2 alleles are paired, but may not be present when 1 or both alleles are paired with other alleles. These conditional ambiguities appear in DPB1 because of lack of polymorphic sites in intron 2. For example, DPB1*04:01 paired with a DPB1*04:02 is ambiguous with DPB1*105:01 and DPB1*126:01 due to loss of phase across intron 2 as the gap between heterozygous positions is too great to phase. However, DPB1*04:01 paired with DPB1*16:01 is unambiguous for both alleles.

What is the turnaround time for TruSight HLA v2?

The turnaround for TruSight HLA from DNA to report is less than 48 hours. Day 1 starts late in the day with long-range PCR taking about 30 minutes to set up for overnight amplification. Day 2 is about 5 hours with about 3.5 hours hands-on time. The sequencer is loaded at the end of Day 2 and takes 17–19 hours to complete depending on the sequencer and the sequencing reagent kit used. Data analysis and reporting on Day 3 requires about 2 hours per 24-sample sequencing run.

Is training required/recommended for TruSight HLA v2?

What training options are available?Training is highly recommended but not required. TR-204-0024 is on-site customer training for TruSight HLA v2 library preparation. Customers receiving on-site library prep training have had a much better experience and have implemented the solution faster than labs not receiving training.

 

Kits

How many samples and loci does the TruSight HLA v2 kit support?

The kit has enough reagents to process 24 samples. TruSight HLA v2 sequences and analyzes HLA-A, -B, -C, -DRB1/3/4/5, -DQB1, -DPB1, -DQA1, and -DPA1.

Is it necessary to sequence all 11 loci for every sample?

We recommend running all the loci on each sample as the pooling post-amplification minimizes the time and cost savings of running fewer loci. If you must run fewer loci, adjust reaction volumes to compensate for the missing loci.

How does TruSight HLA v2 help with keeping pre- and post-PCR workflows physically separate?

TruSight HLA v2 kits include 3 boxes and all pre-PCR reagents are included in a physically separate box (Box 1) from the post-PCR reagents (Boxes 2 and 3).

Where can I find the Safety Data Sheets (SDS) for TruSight HLA v2?

SDS can be found at support.illumina.com/sds. Search for 20000215 (TruSight HLA v2 Sequencing Panel (24 Samples)) or 20005170 (TruSight HLA v2 Sequencing Panel (24 Samples Automated)) for the most current SDS.

How are TruSight HLA v2 reagents shipped?

TruSight HLA v2 Sequencing Panel (24 Samples), catalog # 20000215, includes 3 boxes of reagents. Box 1 includes all the pre-PCR reagents including PCR mix, polymerase, buffers, and PCR primers. Box 1 is shipped on dry ice and is stored frozen at -25°C to -15°C. Box 2 contains purification and normalization beads used post-PCR and is refrigerated at 2°C to 8°C. Box 3 contains post-PCR buffers and tagmentation reagents. Box 3 is shipped on dry ice and is stored frozen at -25°C to -15°C.TruSight HLA v2 Sequencing Panel (24 Samples Automated), catalog # 20005170, includes 4 boxes of reagents. Boxes 1 through 3 are identical to the ones previously described. The fourth box of auxiliary reagents includes additional purification beads required for dead volume minimums on many liquid handlers. The auxiliary reagents are refrigerated at 2°C to 8°C.

What is the minimum shelf-life of TruSight HLA v2 reagents?

TruSight HLA v2 reagents are shipped with a minimum of 3-months of shelf-life.

 

Protocol

What other equipment is needed besides the sequencing platform?

Two thermal cyclers with heated lids per 12 samples, a microplate shaker up to 1800 rpm, a microplate heater is optional (thermal cyclers can be used instead), and 96-well plate magnets for the bead normalization and size selection steps.

What are the safe stopping points in the workflow?

There are 4 safe stopping points in the workflow: (1) after long-range PCR, the amplicons can be held on the thermal cycler at 10°C, (2) after tagmentation clean-up at -20°C, (3) after index PCR at -20°C, and (4) after index PCR clean up at -20°C.

How does TruSight HLA Assign 2.0 detect and identify contamination?

Illumina has designed the workflow to minimize the risk of contamination. However, Assign 2.0 flags any base call that is above the noise but below the minor allele frequency. These flagged positions require visual inspection and it is obvious during this process when contamination has occurred. The sequence reads from the contaminant can be blasted against the NCBI database by right clicking the read, making it easy to investigate the source of the contamination.

How does TruSight HLA v2 tag (ie, index) samples?

TruSight HLA v2 uses a dual indexing protocol using Illumina Nextera XT indexes. Each index is 8 bases in length and the Nextera XT index kits provide i5 and i7 indexes. Each fragmented template has an i5 and an i7 index incorporated. During data analysis, reads with perfect (16 of 16) and one mismatch (15 of 16) are assigned to a sample FASTQ. Libraries with 2 or more mismatches are not assigned to a sample and are not used in the final analysis since these reads are not included in the sample FASTQ file.

How does TruSight HLA v2 purify DNA samples?

The TruSight HLA v2 protocol does not require a purification procedure. However, a bead-based purification method can be used. This method uses SPB (Sample Purification Beads) and RSB (Resuspension Buffer) from the TruSight HLA v2 kit. The TruSight HLA v2 Sequencing Panel (24 Samples Automated) includes an additional tube of SPB that can be used for this purification procedure.
Preparation
1. Bring SPB to room temperature (~30 minutes).2. Prepare fresh 80% EtOH.
Binding
1. Add 50 µl of input sample at 16 ng/µl of high-quality DNA to a 96-well midi plate.2. Add 50 µl SPB to each sample.3. Seal and shake at 1800 rpm for 2 minutes.4. Incubate at room temperature for 2 minutes.
Washing
1. Place on a magnetic stand and wait until beads are fully pelleted and supernatant is clear (~2 minutes). Do not remove from the magnetic stand until instructed to do so.2. With pipettes set to 200 µl, remove and discard 100 µl supernatant.3. Add 200 µl fresh 80% EtOH to each well containing sample.4. Incubate on magnetic stand for 30 seconds.5. Remove and discard all supernatant from each well.6. Repeat EtOH wash.7. With a 20 µl pipette, remove all residual EtOH from each well.8. Air dry on the magnetic stand for 2 minutes.
Elution
1. Add 52.5 µl RSB to each well containing sample.2. Remove from the magnetic stand.3. Seal and shake at 1800 rpm for 2 minutes.4. Incubate at room temperature for 2 minutes.5. Place on the magnetic stand until liquid is clear (~2 minutes).6. Transfer 50 µl supernatant to a new plate.
Quantify using a fluorometric method, such as the Qubit BR assay. Then normalize each sample to 10ng/μl with at least 40 μl RSB per sample.

How does TruSight HLA v2 normalize amplicons and samples?

Bead-based normalization is a technique proprietary to Illumina and is an efficient method for normalization. This method eliminates the need for quantification and any associated equipment that make that process less onerous (eg, plate readers and robots). Bead-based normalization standardizes sample-to-sample and locus-to-locus variability. The bead-based normalization approach allows every amplicon to be normalized individually before pooling. Alternates to this approach involve quantitating every amplicon and normalizing individually based on the quantification. This process is so onerous that most recommend selecting a few amplicons to quantify and then normalizing based on the average. This method is risky as amplification varies from sample to sample and from allele to allele.

What pooling methods does TruSight HLA v2 use?

There are 2 pooling steps in the TruSight HLA v2 workflow. After bead-based normalization and tagmentation, the 8 amplicons (HLA-A, -B, -C, -DRB1/3/4/5, -DQB1, -DPB1, -DQA1, and -DPA1) for a sample are pooled into a single well. The amplicons have been normalized and prepped for index PCR, so combining 10 μl of each amplicon (except DRB, which gets 20 μl) is all that is required.
The second pooling step is the pooling of the samples into a single library for sequencing. 7 μl of each sample is used for quantification using a fluorometric assay. The formula to determine the library volume is 15 divided by the library concentration (in ng/μl). This amount is taken into the dilution and denaturation steps.

What method of fragmentation does TruSight HLA v2 use and what method is used to select appropriate fragment size for sequencing? How can fragment size be measured?

TruSight HLA v2 uses enzymatic fragmentation with a protocol similar to the Illumina Nextera XT protocol. The Nextera technology uses a modified transposition reaction called “tagmentation.” The Nextera transposome enzymes have free DNA ends allowing them to fragment the sample DNA and simultaneously add the sequences required for PCR amplification and sequencing. TruSight HLA v2 uses bead-based size selection to restrict the fragment sizes to greater than 200 bp. TruSight HLA v2 does not require a size selection step to cut off the upper end of the size range as the sequencer will not efficiently cluster fragments over 1.2 Kb.
A user can use a DNA 12000 Bioanalyzer Chip, fragment analyzer, or gel to document these fragment sizes.

How is the specificity of the template in combination with the sequencing primers defined?

Each template is flanked by index primers 8 bases in length, for a total of 16 bases. Only 15 of 16 and 16 of 16 matches are aligned to a sample and used in the analysis. All remaining specificity is managed bioinformatically. Off-target amplification occurs in the presence of certain other HLA genes (eg, HLA-Y and -H) and pseudogenes (eg, HLA-DRB6) and these sequences are identified and removed from the analysis. In addition, off-target amplification between HLA genes (eg, HLA-B primer amplification of HLA-C) can occur in the presence of certain alleles and this data alone cannot be used for reliable typing. Therefore, Assign 2.0 ensures that by assessing primer contribution, HLA-C allele assignment must be from the HLA-C primer amplifications.

What is the specificity of the HLA long-range PCR primers?

Each primer pool (eg, HLA-A, -B, -C, -DRB1/3/4/5, -DQB1, -DPB1, -DQA1, and -DPA1) is designed to account for all known haplotypes for that given locus at the primer site.

Why are HLA-DQB1 amplicons generated using different long-range PCR conditions?

DQB1*06 alleles amplify poorly with the PCR conditions used for the other amplicons. By modifying the PCR conditions, we found the amplification of all DQB1 alleles to be far more uniform resulting in higher confidence typing.

How are PCR crossovers prevented and identified?

PCR cross-over events occur in the amplification of DRB1/3/4/5 when partially amplified products prime subsequent PCR reactions resulting in hybrid amplicons including template from more than one gene (eg, DRB1 and DRB3). On the assay side, we have reduced the number of PCR cycles to 30 cycles in an attempt to reduce the crossovers. However, bioinformatics has been employed to identify and remove any remaining crossovers. Forward and reverse reads are aligned independently to confirm that they match the same target. When bases from within a read can be matched to multiple targets in a sample, those bases are flagged and not used in the final consensus generation. The flagged bases can be observed in the coverage or read views and are marked as 0 quality.

Is there any information on how TruSight HLA v2 performs on FFPE samples?

TruSight HLA uses long-range PCR for isolation and amplification of the HLA genes. These long-range amplicons range in size from 2.8 kb to 10.3 kb. Illumina recommends that the DNA sample consist of at least 50% of the DNA greater than 20 kb because overly fragmented DNA affects the long-range PCR.

 

TruSight HLA v2 Amplicon

Sizes

HLA-A

4.1 kb

HLA-B

2.8 kb

HLA-C

4.2 kb

HLA-DRB1

4.3 kb

HLA-DRB3

4.4 kb

HLA-DRB4

5.1 kb

HLA-DRB5

4.3 kb

HLA-DQB1

7.1 kb

HLA-DPB1

9.7 kb

HLA-DQA1

7.3 kb

HLA-DPA1

10.3 kb

Can I periodically cycle index barcodes?

Yes, TruSight HLA v2 uses the Nextera XT Indexes. 384 unique Nextera XT index combinations are available, so index barcode cycling for runs of fewer than 384 samples is easily achievable.

 

Sequencing

How do I make a sequencing sample sheet for TruSight HLA v2?

We recommend using the TruSight HLA v2 Sample Sheet Template available from the TruSight HLA v2 Support Page. This MS Excel file generates sample names compatible with TruSight HLA Assign 2.0. 

What is the difference in performance between 2 x 250 sequencing and 2 x 150 sequencing with TruSight HLA v2?

We have done extensive testing and found that there is no difference in either typing accuracy or ambiguity rate between 2x150 and 2x250 sequencing with TruSight HLA v2 and Assign 2.0.

We ran 12 TruSight HLA v2 samples on a MiSeq v2 Nano (300 cycles) flow cell with more than enough coverage for each locus. Why do you recommend only 6 samples?

While there is enough coverage on the Nano flow cell for 12 samples for each to achieve 100x mean depth at each locus, optimal library preparation and sequencing conditions are required. If the library preparation or sequencing performance results in lower data output, you may not get sufficient sequencing coverage to achieve 100x mean depth of coverage for every locus.

Is TruSight HLA v2 considered shotgun sequencing assay?

Yes, TruSight HLA v2 is considered a shotgun sequencing approach in which the HLA targets of interest are randomly fragmented, sequenced, and the sequences of the fragments are assembled using bioinformatics.

What metrics can be assessed for measuring reliability of the sequencing data?

We typically measure 3 key metrics for assessing the quality of sequencing run.

Cluster density is measured as K/mm². Cluster density is an important metric that affects run quality, reads passing filter, Q30 scores, and total data output. Underclustering and overclustering are potential problems to monitor. While underclustering maintains high data quality, it results in lower data output and may not produce sufficient reads to achieve optimal depth of coverage. Alternatively, overclustering can lead to poor run performance, lower Q30 scores, possible introduction of sequencing artifacts, and lower total data output due to reads not passing the purity filter. A more detailed review of cluster density can be found in Diagnosing and Preventing Flow Cell Overclustering on the MiSeq System.

Percentage of clusters passing filter (%PF) is an indication of signal purity from each cluster. Overclustered flow cells typically have higher numbers of overlapping clusters, which then causes a decrease is the %PF metric. A low %PF reduces the data output and can result in insufficient reads to achieve optimal depth of sequencing coverage.

Percentage of base calls over Q30 is commonly used to assess sequence quality. A detailed description of quality scores can be found in the Quality Scores for Next-Generation Sequencing Technical Note. Q30 is equivalent to the probability of an incorrect base call 1 in 1,000 times, meaning that the base call accuracy (ie the probability of a correct base call) is 99.9%. When sequencing quality reaches Q30, virtually all the reads are perfect, having 0 errors and ambiguities.

 

Analysis

Is the TruSight HLA Assign software an additional charge?

TruSight HLA Assign software is included with the TruSight HLA v2 kits. The software installer can be downloaded from our website.  If you are a customer and do not have a valid license key, please contact your CareDx representative or Karin Nordenhem at CareDx (knordenhem@caredx.com) who will email a license key to you.

What are the data storage requirements?

Labs typically store zipped FASTQ files (*.fastq.gz) and Assign project files (*.cgp). The total size of all FASTQ files for a run mostly depends on the sequencer and the sequencing flow cell that was used and does not depend on the number of samples.

Flow Cell

Samples

FASTQ File Size

FASTQ File Size Variance

Assign Project File Size

Assign Project File Size Variance

MiSeq v2 Nano (300 cycles)

6

350 MB

+/- 200 MB

100 MB

+/- 50 MB

MiSeq v2 Micro (300 cycles)

12

1.5 GB

+/- 500 MB

200 MB

+/- 50 MB

MiSeq v2 Micro (300 cycles)

24

1.5 GB

+/- 500 MB

350 MB

+/- 75 MB

MiSeq v2 (300 cycles)

48

5 GB

+/- 2 GB

750 MB

+/- 100 MB

MiSeq v2 (300 cycles)

96

5 GB

+/- 2 GB

1.5 GB

+/- 500 MB

What are the recommended computing requirements to run TruSight HLA Assign 2.0?

Minimum Computing Requirements:
· 1 Ghz or faster 64-bit Intel core processor, or equivalent
· 16 GB RAM, minimum
· 16 GB available hard disk space
· Windows OS (Windows Vista, Windows 7, Windows 8, Windows Server 2008, or Windows Server 2012)
· Microsoft Excel 97, or later, for generating reports

How long does it take to process (align, phase, and type) TruSight HLA v2 FASTQ files with TruSight HLA Assign 2.0?

Assign 2.0 does all the processing on the initial load of data. When the data are available for review, the power of the machine does not make a significant difference during review and even machines with minimum specifications perform quickly. The power of the computer has significance in the time it takes to perform the initial processing, which includes importing reads, aligning reads, phasing heterozygote positions, and assigning typing results.

Here are few different configurations and average processing time based on those configurations:

Processor

Memory (RAM)

Flow Cell

Number of Samples

Processing Time

Intel Core i7-5600 2.60 GHz

16 GB

Nano

6

12 minutes

Intel Core i7-5600 2.60 GHz

16 GB

Micro

12

60 minutes

Intel Core i7-5600 2.60 GHz

16 GB

Micro

24

60 minutes

Intel Xeon X5560 2.80 GHz (x2)

96 GB

Nano

6

6 minutes

Intel Xeon X5560 2.80 GHz (x2)

96 GB

Micro

12

25 minutes

Intel Xeon X5560 2.80 GHz (x2)

96 GB

Micro

24

25 minutes

Can I install TruSight HLA Assign 2.0 on a network drive, rather than on individual PCs?

Assign 2.0 can be installed on a network drive, server, or individual PC.  In addition, multiple types of installations can be installed in the same facility.

Does Assign 2.0 require an internet connection?

An internet connection is not required to use Assign 2.0.  However, Assign 2.0 provides links to NCBI Blast, IMGT Allele Database, and AlleleFrequencies.net.  These features require an internet connection.

Does Assign 2.0 run on Mac or Linux systems?

Assign 2.0 is tested and supported on Windows. Virtualization software like Parallels or VMware allows you to run a Windows OS on a Mac or Linux. Therefore, it is possible to run Assign 2.0 through these virtualization technologies. 

How often are IMGT/HLA references updated? Can I update references in Assign 2.0?

IMGT reference updates are made available every 9 months, approximately 2 months after the IMGT release. They are available from the TruSight HLA v2 Support web page.  Follow the instructions provided in the Assign 2.0 software guide to update the references in Assign 2.0. These updates are not performed automatically and can be installed at your convenience. Reference updates do not change the software and the software instance remains unchanged.

Can I use Assign 2.0 to analyze data not generated by TruSight HLA kits or sequencing platforms that are not from Illumina?

Assign 2.0 software was developed to work with data generated using TruSight HLA kits and Illumina instruments. Other sequencing kits and platforms have not been tested and are not supported. Assign 2.0 is compatible with FASTQ files generated from both the TruSight HLA Sequencing Panel (version 1) and TruSight HLA v2 Sequencing Panel.

Can I export sequences from Assign 2.0?

Sample consensus sequences can be exported from Assign 2.0 in MS Excel (*.xls, *.xlsx, or *.xlsm depending on MS Excel version), text (*.txt), or FASTA (*.fasta) formats.

How do TruSight HLA and Assign deal with the intron sequence given that the IMGT/HLA database is missing the intron reference for 90% of the alleles?

Assign 2.0 breaks the sequence for each allele into 3 parts. The Core includes exons 2, 3, and 4 in Class I and exons 2 and 3 in Class II as well as sequence from known expression variants. The second part, Exons, includes all the other exons. By default, users only see sequence and phase mismatches from these 2 parts. It is simple to expand the view to see the third part, N-C, which includes the noncoding UTR and intron sequences. Even though the noncoding sequence is hidden from view by default, heterozygote positions within these regions are used for phase alignment. We also allow users to select the number of fields to review and report (Two, Three, or All).  Ultimately, Illumina is supporting both IMGT and the 17th Workshop to solve the root of the issue, which is the missing noncoding reference.  

Does Assign 2.0 exclude primer sequences from consideration in the assignment of alleles?

Yes, the primer sequences are excluded from the analysis and allele assignment.

What criteria does Assign 2.0 use to establish an allele assignment?

Assign 2.0 uses a perfect match approach to assigning alleles. To be assigned, the sample consensus sequence (consensus of all reads used at each base position) must match an IMGT/HLA exactly (both base call and phase). Even 1 mismatch in base call or phase results in no allele assignment. When there is a mismatch, the only way to make an allele assignment is to edit the sequence, if warranted. These edits are tracked and auditable.

What bioinformatics processes does Assign 2.0 use to process sequence data?

Assign 2.0 first performs alignment of the sequencing reads. The alignment is performed against a locus consensus sequence generated from the alleles for each locus. The heterozygous positions of these aligned reads are then phased. The first pass of phasing phases heterozygous positions within the same read. The second pass of phasing phases heterozygous positions fulling within the same read pair. The final phasing step layers paired reads to determine phase between heterozygous positions for which the first 2 passes were unable to elucidate phase. If all 3 of these passes fail to determine phase or if the data provides ambiguous phasing, phase is not assigned and a phase break is shown.
These phased alignments are then compared to the IMGT/HLA database within Assign 2.0 and can be assigned an allele, multiple alleles, or no alleles. If an unambiguous alignment to a single allele is made, then this result is displayed on the summary report. If the result is ambiguous (multiple perfect matches), all perfect matches appear in the report. If no perfect match is available, these receive a no call and require manual review and editing, if warranted.

What steps are taken to prevent and identify PCR artifacts?

PCR artifacts are common when amplifying gene targets. Artifacts are known to occur in the TruSight HLA v2 assay, but it has been optimized to amplify them at a very low rate. Furthermore, Assign 2.0 provides visualization of the sequencing reads to assess read diversity. Assign 2.0 is also designed to seek read diversity and uses a broad range of reads, reducing the likelihood of PCR artifacts contributing to the allele assignment.

Can multiple users look at the same data simultaneously and independently in Assign 2.0?

Assign 2.0 can be installed on a shared server or network drive, and multiple users can independently launch and simultaneously use the same instance of Assign 2.0. These instances share only the settings file and the data under review can be from the same sample. However, edits, comments, and user-defined settings are not available between users.

How is contamination identified and measured?


Contamination appears as additional alleles in the results. Assign 2.0 calls the 2 most frequent alleles in the sample. All other base calls are either flagged or considered noise if they fall within the acceptable range. Base calls are flagged because they are higher than the noise, but have not achieved the frequency of the second most frequent base in the locus. Review flagged bases to quickly check whether contamination is present and to measure the degree present.
Furthermore, reads from these bases are available in the viewer. Right-click to blast these reads against the NCBI database to help determine the likely source of contamination.

Can I edit a sequence in Assign 2.0 and can the edit be viewed in the report?

Yes, sequences can be edited using the Navigator functionality. Edits are flagged in the Coverage View and can be found using the forward and reverse arrows in the Navigator. There is an option to include sequence edits on the report, which shows the edit, base position, and user.

What file extensions does Assign 2.0 generate?

Assign 2.0 has 4 output options:

Full Report—Shows all selected loci and samples with all perfect matches with the option to include the edits, sequences, and mismatches. Available MS Excel (*.xls, *.xlsx, or *.xlsm), text (*.txt), and XML (*.xml).

Summary Report—Shows only unambiguous typings for all the samples in the project, quality scores for each locus, and coverage for each locus. Available as MS Excel (*.xls, *.xlsx, or *.xlsm) and text (*.txt).

Project Files—Allows the project to be reloaded without reanalysis (alignment, phasing, and typing). Output as *.cgp, an XML-based file specific to Assign 2.0.

FASTA—Output as *.fasta.

Is Assign 2.0 an upgrade or new installation?

Assign 2.0 is a new installation and can be run side-by-side with Assign 1.0 for TruSight HLA.

Can Assign 1.0 Project Files (*.cgp) be imported into Assign 2.0?

Assign 1.0 Project Files (*.cgp) are not compatible with Assign 2.0. However, Assign 2.0 can import and analyze FASTQ files generated by TruSight HLA Sequencing Panel (version 1).

How does licensing work and how long is my software license valid?

Assign TruSight HLA software is provided free-of-charge to TruSight HLA customers. At the end of each licence period you will receive a new license key(s) via email. The license can be used for an unlimited number of samples, unlimited number of systems, and unlimited number of users. After the key has expired, previously saved projects (*.cgp) can be analyzed without an active license but new FASTQ files cannot be imported. To request a new key, contact your CareDx representative or Karin Nordenhem, marketing support (knordenhem@caredx.com).