Frequently Asked Questions About Sequencing
Below you will find some of the questions that we are frequently asked concerning sequencing. If you have a question that is not listed below, please contact us.
What are the basic differences between using Illumina and Roche sequencing?
The Illumina HiSeq platform provides many reads, and therefore, more samples can be pooled per lane than Roche 454. Overall, Illumina sequencing is more cost effective than Roche 454. The read length for Illumina HiSeq is approximately 150 base pairs and the Illumina MiSeq and Roche 454 are approximately 250 base pairs. Therefore, the Illumina platform is recommended for TCR sequencing and provides CDR3 data.
Roche 454 and Illumina MiSeq sequencing is recommended for B cell studies because the read length is longer, approximately 350 bp with sequence information beginning from within the first framework region and extending into the beginning of the C-region. The trade-off for increased sequencing length with Roche 454 is reduced sequencing depth (ie. fewer available reads) when compared to using the Illumina system. In addition, Roche 454 sequencing is more expensive than Illumina, and fewer samples should be pooled per run (due to the reduced availability of reads). However, if you are expecting your library to lack diversity (ie. clonal expansion of just a few clones), then pooling more samples is possible. The longer read length of the 454 and MiSeq platforms allow for the determination of the class of the antibody and hypermutation information.
Are the Illumina libraries produced by iRepertoire’s primers compatible with paired-end sequencing, or are they restricted to single-end reads?
The Illumina libraries produced are compatible with paired-end sequencing. In fact, for our Illumina systems, we recommend paired-end sequencing because paired-end reads are from one DNA fragment. This information is useful for extending sequencing reads to cover the entire CDR3 region and for identifying V and J germline segments reliably. In addition, we also use the paired-end information to calibrate whether a CDR3 fragment is authentic.
After the second round of amplification with iRepertoire’s primers, the full-length Illumina sequencing adaptor A (below) is associated with an amplicon at the C or J region (primer system dependent) while adaptor B (below) is associated with an amplicon at the V region.
After the size-selection procedure, the purified product should go though cluster generation directly without further amplification as the full-length Illumina pair-end sequencing adaptors have been attached to the V(D)J sequences already.
Are the Roche 454 libraries produced by iRepertoire’s primers compatible with paired-end sequencing, or are they restricted to single-end reads?
The Roche 454 libraries produced with iRepertoire’s primers are restricted to single-end reads from primer A using the Roche 454 Lib-A sequencing kit.
How many samples can we pool together?
You can pool up to 40 (60 for Human TCR beta) individually barcoded samples per lane with the Illumina platform. For the Illumina MiSeq platform, we have up to 10 individual barcodes for pooling samples. However, for 454 sequencing, we do not recommend pooling more than 5 samples per sequencing run (full plate). The number of samples pooled per lane (or plate) is dependent on the desired depth of sequencing.
We were able to obtain a library after gel extraction but the amount is more than 500 ng/library. Is this acceptable?
Normally, we pool 20 - 40 libraries together for Illumina HiSeq sequencing. 500 ng total of pooled library (ie. more than one library if you plan to sequence more than one sample at the same time) is enough for sequencing, but we prefer to have at least 1 μg pooled library for a possible repeat experiment at a recommended concentration of at least 20 ng/μl. You should pool your libraries to fit this recommendation. If the concentration of the pooled libraries is too low, we recommend using a PCR purification kit to concentrate the sample by repeatedly applying the sample to the same column and eluting in a smaller volume.
Concerning the sequencing depth in repertoire analysis of small population Ag-specific T cells, can we sequence their repertoire with lower depth, like 50-100 barcoded samples in a lane?
We provide up to 60 barcodes for both the HTBIvc and the HTAIvc systems (RNA only). We currently offer 20 barcodes for the HTBIvj reagent system (RNA and gDNA).
How much DNA/RNA is needed for sequencing, the more the better?
This question is really two-fold. First, one might ask what is the minimum amount of starting template to obtain a diverse library? A minimum of 100 ng of RNA or gDNA with a 260/280 of 1.8 or greater is recommended as the starting material to obtain the library. However, the addition of more template will likely increase the diversity of the library. Please see “Sample Requirements” for further information. Ultimately, the appropriate amount of template added to the reaction must be determined empirically. The maximum input RNA of the Qiagen One-Step RT-PCR kit is ≤ 1µg per 25 µL reaction.
The second part of the question is then how much pooled library do I need for sequencing? After pooling samples, we recommend that you send us 1ug of pooled library at a minimum concentration of 20ng/ul (see “Sample Requirements”). If you do not choose us for sequencing, please refer to your sequencing vendor for their requirements.
Can we enlarge the final volume of the library to be sequenced to 100 μL to obtain more DNA, but the concentration might be lower than 10 ng/μL?
The volume can be more than 100 μL, but we prefer the concentration to be greater than 20 ng/μL. To avoid a pooled library with too low of a concentration, it is recommended to elute the gel purified product of each sample in a smaller volume prior to pooling (ie. increase the concentration of each sample). Alternatively, you could concentrate the pooled library.
Are there specific requirements for the DNA library sample to be sequenced, like A260/230 ratio?
Typically, we require the concentration measurements listed above, and A260/280 ratio of at least 1.8 (higher is fine). A high quality extraction kit should provide these results without issue if used according to the manufacturer’s recommendations.
Can we store the DNA library in EB buffer provided by Qiagen Gel Extraction kit for subsequent sequencing?
We are now using Ethidium Bromide for DNA staining during gel extraction. For safety consideration, we want to switch that dye to Gel-red, will it affect the subsequent sequencing? What DNA dye do you use in your laboratory during gel extraction?
We use Ethidium Bromide in our lab. We don’t have experience using Gel-red; however, we do not anticipate that this would be an issue.