152 Reference | Support Invivoscribe 2019 | 153 Reference Support message may be generated. Furthermore, the software is only compatible with adaptor-trimmed fastq.gz files that are generated by the MiSeq® Reporter Software when the MiSeq® instrument is used. An example of the naming format that the MiSeq® Reporter uses: SampleName_S1_L001_R1_001.fastq.gz and SampleName_S1_L001_R2_001.fastq.gz. 25.  Do the Invivoscribe MiSeq® indices correspond to the Illumina® indices? The indices included in our MiSeq® master mixes follow Illumina® ’s TruSeq LT nomenclature. For instance, IGH FR1 MiSeq® 01 corresponds to A001. Information for the other indices can be found in the instructions for use on how to set up the MiSeq® Sample Sheet to detect the appropriate indices. 26.  Why am I getting a low percent passing filter and Q30 score? Low Q30 and percent passing filter (%PF) scores could be an indication that the flow cell is overloaded. If this is suspected, verify your amplicon and library calculations and quantifications are correct. Low run metrics can also be attributed to many additional factors including poor quality DNA, contamination, flow cell or instrument issues, etc. Please refer to your Illumina MiSeq® user guides and contact Illumina® Support. 27.  Why is the same VH-JH rearrangement combination and sequence shared by two groups of reads, one of which is several bases shorter than the other when looking at the Read Summary tab of the excel document created by the LymphoTrack® Visualization Tool? Our software was designed to list every unique sequence separately in order for the customer to see all of the data and make their own determination on how to interpret it. The several base pair difference can be due to a number of factors including amplification errors and sequencing errors. It could also be a result of similarities between some of the primer sequences that were designed to ensure maximum coverage. We also include a Merged Read Summary report for your reference that combines sequences that only differ by 1 or 2 basepairs. 28.  Do I need to perform an adapter ligation prior to sequencing my products? Performing an adapter ligation is not needed. The primers included in our LymphoTrack and LymphoTrack Dx master mixes already include the appropriate index barcodes and adapter sequences. After PCR amplification, you will be able to proceed with amplicon purification, amplicon quantification, library pooling, and sequencing. 29.  If the LymphoTrack® or LymphoTrack® Dx software generated an error, what information should I submit to Technical Support? Please submit the *.txt Log file that should have been created by the software in the output folder, a screenshot of the sample directory, and the Lot Number of the software CD you are using to support@invivoscribe.com. 30.  Are controls provided with the kits? Can you purchase additional controls? How are they supplied? Each kit contains the necessary positive and negative controls required to perform the assay; additional controls may also be purchased separately. Single-tube DNA controls are provided as 100 μL aliquots of 200 μg/mL in 1/10 TE Buffer, 50 μL aliquots of 50 μg/mL in 1/10 TE Buffer, and 45 μL aliquots of 15 μg/mL in 1/10 TE Buffer. Single-tube RNA controls are provided as 100 μL aliquots of 400 μg/ml in RNAse free in glass distilled water. 31.  What are the differences between dilution sets, sensitivity panels, and proficiency panels? 31a.  RNA Dilution Sets BCR/ABL b3a2 (Cat# 4-085-0210), BCR/ABL b2a2 (Cat# 4-085-0310), and BCR/ABL e1a2 (Cat# 4-085-0110). These sets contain six tubes: 100% negative control RNA and volume to volume (v/v) dilutions (10-1 , 10-2 , 10-3 , 10-4 , and 10-5 ) of the positive control RNA into the negative control RNA (IVS-0048). The RNA Dilution Sets are supplied at a concentration of 400 μg/mL, and each tube contains 50 μL. These dilution sets may be used to establish a standard reference curve, as proficiency controls, as sensitivity controls for specific target assays, and as routine testing controls for cDNA synthesis, amplification and detection. 31b.  RNA Sensitivity Panels These panels consist of seven tubes: 100% positive control RNA and v/v dilutions (10-1 , 10-2 , 10-3 , 10-4 , 10-5 , and 10-6 ) of the positive control RNA into the negative control RNA (IVS-0035). The RNA Sensitivity Panels are supplied at a concentration of 400 μg/mL, and each tube contains 100 μL. The RNA Sensitivity Panels may be used as sensitivity controls for specific target assays, and as routine testing controls for cDNA synthesis, amplification and detection. 31c.  DNA Sensitivity Panels Consist of six tubes: 100% clonal DNA and v/v dilutions of the clonal DNA into negative polyclonal DNA (IVS-0000) to make 30%, 20%, 10%, 5%, and 1% dilutions. The DNA Sensitivity Panels are supplied at a concentration of 200 μg/mL and each tube contains 100 μL. The DNA Sensitivity Panels may be used as sensitivity controls for specific target assays. 31d.  RNA Proficiency Panel The proficiency panel for BCR-ABL1 t(9;22) can be used as a sensitivity control for specific target assays, and as routine testing controls for cDNA synthesis, amplification and detection. It consists of ten tubes: 100% positive control RNA and v/v dilutions (10-2 and 10-4 ) of IVS-0003, IVS-0011 and IVS-0032. It also includes BCR-ABL1 Negative Clonal Control RNA (IVS-0035). generated. The T-Cell Receptor Gamma Gene Rearrangement Assay 2.0 was designed by Invivoscribe and targets all functional VH - JH rearrangements in a single master mix and produces smaller amplicons grouped under a single Gaussian distribution. This allows for easier interpretation and makes the assay more suitable for DNA extracted from FFPE tissue, which may consist of partially degraded DNA that would not amplify well with the larger valid size range of the TCRG Gene Clonality Assay. 12.  What are the differences between the IGH-BCL2 Translocation Assay and the IGH-BCL2 t(14;18) Translocation Assay? The IGH-BCL2 Translocation Assay was designed by the EuroClonality/BIOMED-2 Group and is available as either a CE-IVD or research use only assay whereas; the IGH-BCL2 t(14;18) Translocation Assay was designed by Invivoscribe and is only available for research use only. Both of these assays target MBR and Mcr translocations, but the IGH-BCL2 Translocation Assay also targets the translocations at the 3’ Mbr. The IGH-BCL2 t(14;18) Translocation Assay was designed as a nested PCR allowing greater sensitivities (1 clonal cell per 10,000 normal cells) to be achieved. The limit of detection of the IGH-BCL2 Translocation Assay is 1 clonal cell per 100 normal cells. Lastly, the IGH-BCL2 Translocation Assay includes 33 reactions, whereas the IGH-BCL2 t(14;18) Translocation Assay includes 30 reactions. 13.  Do you offer quantitative chromosome translocation (e.g., BCR-ABL1) controls? Our controls are validated for qualitative use, although our customers do successfully use them with quantitative assays. Unfortunately, we cannot guarantee their performance with any assay that was not designed by Invivoscribe. 14.  Which capillary electrophoresis instruments are currently validated for use with our assay kits? Currently the capillary electrophoresis instruments Invivoscribe has validated include: ABI 3100 and 3130 series for all capillary electrophoresis detection assays. The ABI 310 and 3500 instrument series have also been validated for the majority of our capillary electrophoresis detection assays. We are not able to support using instruments not listed as validated in the instructions for use of our CE-IVD assays. 15.  What are the recommended settings for my ABI instrument? Instruments should be calibrated with the DS-30 matrix standards (Dye set D) for the ABI 310, 3100, or 3130 instrument series. For the ABI 3500 sequencer series, we advise that you calibrate the instrument with DS-33 matrix standards. We also recommend using either POP-4 or POP-7 depending on which ABI instrument you are using. If your equipment supports POP-7, we recommend using this polymer as it can be utilized for both fragment analysis and sequencing; whereas, POP-4 can only be utilized for fragment analysis. 16.  How should peaks outside the valid size range be interpreted when using assay kits? You should not interpret peaks outside of the valid size range; although, in theory, it is possible to have a true rearrangement fall outside this region. If you are concerned about a suspect peak, you may sequence your product for confirmation. Please note that samples should always be interpreted within the context of all available clinical information. 17.  Is cell-free DNA (cfDNA) a suitable sample type for Invivoscribe LymphoTrack® or LymphoTrack® Dx Assays? The average size of cfDNA (~170 bps) makes it a suitable sample type to run with IGH FR3 master mixes. The use of cfDNA with TRG master mixes might be possible, but expected amplicon sizes generated with this assay are near the upper limits of the fragment lengths typically found with this sample type. 18.  Is DNA extracted from FFPE tissue suitable to use with Invivoscribe LymphoTrack® or LymphoTrack® Dx Assays? To ensure DNA from challenging specimens is of sufficient quality and quantity to generate a valid result, samples may be tested with the Specimen Control Size Ladder master mix. 19.  On which instruments can I use the LymphoTrack® and LymphoTrack® Dx Assays? We have different versions of our assays for the PGM™ and MiSeq® instruments (LymphoTrack TRB is currently available only on MiSeq® ). No other DNA sequencers (e.g. 454) are currently supported. Assays for the Ion PGM™ and MiSeq® platforms differ slightly in terms of the total number of indices, etc., but both have similar benefits such as a one-step PCR reaction and included bioinformatics software. 20.  How much DNA is needed for the LymphoTrack® and LymphoTrack® Dx Assays? 50 ng of high-quality genomic DNA is required for the Ion PGM™ and MiSeq® LymphoTrack and LymphoTrack Dx Assays for clonality and somatic hypermutation applications. 21.  Can I use a different library quantification method or kit? We recommend using the KAPA™ kit for MiSeq® assays and either the 2100 Bioanalyzer® or the LabChip® GX for the Ion PGM™ assays. 22.  Will the LymphoTrack® or LymphoTrack® Dx analysis software work on my computer? The software requires Microsoft Windows 7 (64-bit) and Excel 2007, 2010, or 2013 and will work with most desktop or laptop PCs. For specific requirements please refer to the software instructions for use. 23.  Can I use the LymphoTrack® or LymphoTrack® Dx bioinformatics software with a different assay? No, the software will only work with datasets obtained by our LymphoTrack and LymphoTrack Dx Assays. 24.  What characters can I use when naming my samples and the file pathways? What types of files are accepted by the LymphoTrack® and LymphoTrack® Dx Software - MiSeq® ? Our software only recognizes file names and pathways that contain the following characters (A-Z, a-z, 0-9, . (dot), _ (underscore), - (hyphen)). In addition, spaces in the pathname for the data files or software (pathnames include file folders and file names) should be avoided. If the software encounters a character that is not listed above or extra spaces, an error