The basics of single stranded DNA NGS Kits

Q: How is the SRSLY Kit able to capture dsDNA molecules if all DNA molecules are denatured and kept as ssDNA throughout the whole protocol?

A.  Both strands of the dsDNA exist as independent ssDNA molecules after denaturation. During the SRSLY ligation reaction both molecules from the original duplex DNA have an equal but independent probability of receiving NGS adapters during ligation. SRSLY (nor any other ssDNA based library prep method) does not have the ability to perform duplex aware sequencing due to its single-stranded nature and the fact that it retains native 5’ and 3’ termini.

Q: Why does the SRSLY Kit capture nicked, fragmented, and damage DNA molecules better than standard dsDNA prep kits?

A: Unlike the SRSLY protocol, dsDNA prep kits maintain the DNA as duplex DNA through adapter ligation and up until initial denaturation during index PCR. If a dsDNA library molecule contains heat labile damage or one of the duplex DNA strands is nicked, the heat during initial index PCR denaturation will cause a break in that particular strand of the DNA. When that strand breaks, it is lost to sequencing since it no longer contains adapters on both ends of the broken strand. In contrast, SRSLY performs an initial heat denaturation step before adapter ligation. Heat labile and nicked strands are broken during this step and then all molecules are subsequently ligated with adapters before index PCR.

SRSLY Specific kit faqs

PicoPlus vs NanOPLus

Q: What is the difference between the PicoPlus and the NanoPlus kits?

A: The structure, composition, and concentration of the adapters differs between the two kits. The PicoPlus kit works best with DNA inputs less than 10 ng and is a great choice when sample DNA is limiting. The NanoPlus kit works the best with DNA inputs greater than 10 ng and is a great choice when sample DNA is not limiting.

Customers now have the option to purchase either our standard index PCR polymerase or an uracil tolerant index PCR polymerase when purchasing a SRSLY base kits (Pico/Nano). Customers should consider purchasing the uracil tolerant index PCR polymerase containing base kit (Pico/Nano) when their template DNA contains uracils. Some example templates are as aDNA, some FFPE samples and methyl-converted DNA. Customers working with all other template DNA types should utilize the SRSLY kit containing the standard index PCR polymerase. Customers should purchase the SRSLY kit containing the standard index PCR polymerase when purchasing the UMI add-on kit regardless of template DNA type because the UMI extension polymerase is uracil tolerant. Therefore, there is no need for an uracil tolerant index PCR polymerase.

srsly overview

Q: How exactly does SRSLY work?

A: Up to 18 µl of template DNA is mixed with our single-stranded enhancer protein and then put through a quick cold-heat-cold shock. Afterwards the reaction is brought up to 50 µl with SRSLY adapters and ligation/phosphorylation master mix and then incubated for 1 hr. The adapters are directional and blocked on all ends except for the termini that is required for proper NGS library prep. The SRSLY master mix contains a 5’ kinase, a 3’ phosphatase, and a ligase. The phosphatase and kinase prepare the termini for ligation without modification to the bases on the 3’ termini. After the 1 hr incubation, samples are bead purified and then placed into index PCR. After index PCR and a final bead purification, libraries are ready for quantification. Below is a schematic of the overall protocol. For details please check out the SRSLY methods paper published in BMC Genomics in 2019.

srsly protocol

Q: Can I see the SRSLY protocol manual?

A: Unfortunately the full unabridged protocol is only available to customers post purchase. Those interested in the SRSLY protocol basics can contact customer support (technicalsupport@claretbio.com) to receive a copy of the abridged quick start protocol.

SRSLY UDI Primer faqs

Q: What are the sequences and concentrations of your UDI primers?

A: Forward Primer Sequence (i5): Supplied at 5 µM in the pooled primer tube or well;
5′-AATGATACGGCGACCACCGAGATCTACACXXXXXXXXACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’

Reverse Primer Sequence (i7): Supplied at 5 µM in the pooled primer tube or well;
5′-CAAGCAGAAGACGGCATACGAGATXXXXXXXXGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’

Notes: The X’s in the primer sequences correspond to the bases in the table below. The UD-24 contains 2 reactions worth of each of the first 12 i5/i7 UDI combinations of the UD-96 plate in a 12-tube format.

Q: How many UDI primers do you offer?

A: We currently only offer up to 96 UDI’s. If additional UDIs are desired, the NEBNext Multiplex Oligos for Illumina are fully compatible with SRSLY (NEB Cat No. E6440S/L).

SRSLY UMI Primer FAqs

Q: What’s the deal with UMIs and SRSLY?

A: UMIs or Unique Molecular Indexes are molecular barcodes useful for detecting unique molecules, SNPs, and resolving PCR duplicates. The UMI addition module for SRSLY (UR- ) provides users a robust solution to add UMIs to any library prepared with the SRLSY Kit. The UMI kit is not required to create SRSLY libraries.

Q: How exactly does the UMI addition module work with SRSLY?

A: The UMI addition step (performed prior to index PCR) tags a random 9 nt UMI to every SRSLY adapter ligated molecule via primer extension. This optional step adds a 30 minute primer extension step and a 30 min bead purification step to the overall protocol. After cleanup the primers for index PCR differ than those for non-UMI SRSLY as the index PCR primers used subsequently (CBS-UM-24, CBS-UM-96 or CBS-UM-24-4 ) only amplify the UMI-containing strand. Below is a figure detailing the UMI addition step.

Q: What are the sequences and concentrations of your UMI primers?

A: i7 Extension Primer Sequence (i7 ext): Supplied at 5.3 µM;
5′-CAAGCAGAAGACGGCATACGAGATNNNNNNNNNXXXXXXXXGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’

Forward Index Primer Sequence (i5): Supplied at 5 µM in the tube with Ui7;
5′-AATGATACGGCGACCACCGAGATCTACACXXXXXXXXACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’

Reverse Index Primer Sequence (Ui7): Supplied at 5 µM in the tube with the i5 index;
5′- CAAGCAGAAGACGGCATACGA-3’

Q: How many UMI primers do you offer?

A: We currently offer up to 96 UMI-UDI primers. The 96 unique i7 Extension Primers are contained in 96 well plate 1 of 2 of the UM-96 catalog item. The 96 unique i5 indexes are contained in the 96 well plate 2 of 2 of the UM-96 catalog item. The i5 index plate is premixed with the Ui7 primer in each well of the plate.

Q: Can I purchase additional UMI primers from a third party?

A: Unfortunately there are no third party options for SRSLY compatible UMI primers. The design is unique to Claret Bio and as such we are the only supplier. We have custom options for researchers interested in expanding their UMI primer diversity. Email technicalsupport@claretbio.com for more information.

Q: The UMI is in the i7 index read, how does that work?

A: There are special considerations that need to be taken when setting up a sequencing run so that the UMI bases are captured in the i7 index read. First, the number of bases allocated for the sequencing of the i7 index read needs to be increased from 8 to 17. Second, researchers need access to the raw bcl files, not the fastq files. If unfamiliar with the steps necessary to properly sequence the UMI bases, please contact us at technicalsupport@claretbio.com or contact Illumina technical support.

In order to properly demultiplex UMI data post sequencing, we offer an open-source bioinformatic solution. Please check out our Software Page for more information.

clarefy bead faqs

Q: What exactly are the Clarefy beads?

A: The Clarefy beads are Claret Bio’s formula of DNA purification beads. They look and function similar to all other DNA purification beads

Q: Can I use other DNA purification beads or do I have to use Clarefy beads with SRSLY?

A: Although beads from different manufacturers behave slightly differently, all DNA purification beads work well with SRSLY. The specific Clarefy beads are not required to perform SRSLY. We have thoroughly vetted other beads in conjunction with SRSLY and detail the slight differences in use between Clarefy beads, AMPure beads (Beckman Coulter), and Zymo’s Select-a-Size DNA MagBeads in Appendix C of our Unabridged Manual. You can read our application note for comparison between different DNA purification methods.

Q: Which of SRSLY’s three DNA bead purification options is right for my sample?

A: DNA templates come in all shapes and sizes. Choosing the correct bead purification option for your sample will increase library product and decrease adapter dimers and read discarded. The below table and decision tree should help you decide which of the three bead purification options is right for you.

Input DNA faqs

srsly cell free DNA faqs

Q: What are your recommendations for preparing cfDNA from blood plasma for SRSLY?

A: For storage and extraction of blood we recommend using the Cell-Free DNA BCT blood collection tube from Streck (Streck Cat. No. 218961, 218962, 218992). For an extraction kit we recommend using the QIAGEN QIAamp MinElute ccfDNA Kit for cfDNA Extraction (Qiagen Cat No. 55204).

For an extraction protocol we recommend:

1. Spin blood collection tubes at 1800 g for 10 minutes at 4°C in a temperature-controlled tabletop centrifuge.
2. Without disturbing the cell layer (buffy coat), transfer supernatant (plasma) in 2 ml aliquots to 2 ml microfuge tubes.
3. Spin 2 ml microfuge tubes at 16000 g for 10 minutes at 4°C to remove cell debris.
4. Transfer supernatant to new 2 ml microfuge tubes.
5. Extract cfDNA from 4 ml of twice spun plasma using the QIAGEN® QIAamp® MinElute® ccfDNA Kit for cfDNA extraction.

Q: What do you define as “high quality cfDNA”?

A: Typically, “high quality” cfDNA samples contain a large mono-nucleosome peak, centered around 140 – 180 bp (depending on the imager used) and a much smaller di-nucleosome peak centered around 320 – 360 bp. Rarely, but occasionally “high quality” cfDNA samples will also contain an observable tri-nucleosome peak around 510 bp. Observable DNA higher than 510 bp usually denotes cellular genomic DNA contamination within the cfDNA extract. Here is an example of “high quality” cfDNA:

srsly FFPE DNA faqs

Q: What are your recommendations for preparing FFPE DNA for SRSLY?

A: We recommend FFPE DNA be extracted using the Qiagen QIAamp DNA FFPE Tissue Kit (Qiagen Cat No. 56404) and manufacturer’s instructions, or a kit of similar quality. The purchase and use of Xylenes is required to melt the paraffin. The use of a fume hood is highly recommended.

Post DNA extraction we recommend FFPE extracted DNA to be quantified post extraction for concentration using the Qubit dsDNA HS assay kit and manufacturer’s instructions.

Since FFPE DNA is fragmented but variable in length we recommend assaying the length distribution of extracted FFPE DNA to determine if FFPE DNA needs to be sheared prior to sequencing library preparation. Length distribution analysis can be performed with either an Agilent TapeStation, Bioanalyzer, or Fragment Analyzer. In our experience, FFPE-extracted DNA with an average size distribution greater than 700 bp should be sheared prior to the SRSLY protocol in order to be confident that the maximum number of molecules possible are within the correct size range for sequencing. While extracted FFPE DNA shorter than 700 bp does not technically require shearing, we have observed increased mapping and complexity rates when shearing is performed. We recommend shearing DNA to a normal distribution centered somewhere between 400 - 500 bp in size using an acoustic sonicator. See the trace in the genomic DNA section for a visualization of a normal distribution.

srsly genomic dna faqs

Q: What are your recommendations for preparing gDNA for SRSLY?

A: Genomic DNA extraction techniques vary widely depending on the Kingdom and Phylum of the organism. Please follow the appropriate standard protocols for gDNA extraction from your study organism. 

Post DNA extraction we recommend extracted gDNA to be quantified post extraction for concentration using the Qubit dsDNA HS assay kit and manufacturer’s instructions.

Genomic DNA is generally high molecular weight. Input gDNA must be fragmented prior to SRSLY. We recommend shearing DNA to a normal distribution centered somewhere between 250 – 400 bp in size using an acoustic sonicator. Here is an example of suitably sheared DNA:

SRSLy RNA-seq faqs

Q: Can I use SRSLY for RNA-Seq?

A: In order to prepare cDNA for input into SRSLY, purified RNA must usually be depleted of rRNA or enriched for the desired RNA, then reverse transcribed and purified prior to SRSLY. We now have a new bundle that includes all the components for RNA-Seq - first-strand synthesis, ribo-depletion and library preparation - called REALLY-rNONE. Learn more here. Alternatively, you could also use the Zymo-Seq RiboFree Universal cDNA Kit (Zymo Cat. No. R3001) upstream of SRSLY. Another option we recommend for total RNA inputs upstream of SRSLY are New England Biolabs suite of RNA enrichment and depletion products as well as their NEBNext Ultra II RNA First Strand Synthesis module (NEB Cat No. E7771) pre-SRSLY.

srsly oligo dna faqs

Q: What oligos work as input for SRSLY?

A: Oligos purified by any means (standard desalt, PAGE, HPLC) work equally well as input for SRSLY. Oligos being assayed must be free of both ligation and extension blocking modifications. Oligos containing blocking modifications other than a 3’ Phosphate will not work as input into SRSLY. Oligo’s containing a 3’ Phosphate must be prepared in a certain way to maximize complexity (contact technicalsupport@claretbio.com for details). Due to the sequencing restrictions of Illumina machines oligos larger than about 800 nt must be sheared prior to SRSLY input. You can find information about SRSLY’s utility in oligo QC here.

srsly methyl-seq faqs

Q: What are your recommendations for a Methyl-Seq powered SRSLY?

A: Prior to methyl conversion we recommend that high molecular weight DNA inputs be sheared to a normal distribution with a mean of approximately 500 bp via sonication or enzymatic means. For enzymatic shearing we recommend the NEBNext dsDNA Fragmentase. After shearing we recommend the DNA be quantified for concentration using the Qubit dsDNA HS assay kit and quantified for length distribution using an Agilent TapeStation, Bioanalyzer, or Fragment Analyzer.

Post DNA quantification we recommend that the input DNA be methyl-converted using either the Zymo EZ DNA Methylation-Lightning Kits (Zymo Cat. No. D5030, D5032, D5033, D5046) or the NEBNext Enzymatic Methyl-seq Conversion Module (NEB Cat. No. E7125S, E7125L). Both kits when utilized upstream of SRSLY produce excellent results. Simply elute the final clean of either kit in 18 µl and use all of the purified DNA as input into SRSLY.

Customers should purchase the uracil tolerant index PCR polymerase containing SRSLY kit (Pico/Nano) when working with methyl-converted DNA without UMIs. Customers should purchase the standard index PCR polymerase containing SRSLY kit (Pico/Nano) when working with methyl-converted DNA with the UMI Add On Reagent. This is because the UMI extension polymerase is uracil tolerant and therefore there is no need for an uracil tolerant polymerase at the index PCR step.

srsly deamination faqs

Q: What do I do if my input DNA sample has high amounts of deamination (Uracil’s)?

A: Customers should purchase the uracil tolerant index PCR polymerase containing SRSLY kit (Pico/Nano) of their choice when working with DNA templates containing higher amount of uracils and do not require the UMI Add On Reagents. Customers should purchase the standard index PCR polymerase containing SRSLY kit (Pico/Nano) when working with DNA templates containing higher amount of uracils in conjunction with the UMI Add On Reagent. This is because the UMI extension polymerase is uracil tolerant and therefore there is no need for an uracil tolerant polymerase at the index PCR step.

SRSLY Shearing faqs

Q: Above it is mentioned that FFPE DNA, gDNA, and methyl converted DNA all must be sheared to a normal distribution prior to SRSLY but the shearing sizes you mentioned are different, why?

A: All high molecular weight DNA must be sheared to be compatible with Illumina sequencers due to the specifics of bridge amplification. As long as high molecular weight DNA of any input type is sheared to a normal bell curve distribution centered around any size of about 500 bp or less SRSLY, and sequencing, will work well.

As for the specifics of the different sizes, these three input types (gDNA, FFPE DNA, methyl converted DNA) behave slightly differently within SRSLY. Genomic DNA is high quality DNA that is free of DNA damage. As such, the shearing distribution should reflect the insert size of the library post sequencing. However, that is not the case for FFPE DNA and methyl converted DNA. Both FFPE DNA and methyl converted DNA are damaged. As such, the final library size and insert length for these input types are usually less than the initial shearing profile. Therefore, if longer insert lengths are desired for these two input types, it benefits the user to initially shear these input types to a slightly larger size knowing that the final libraries will be shorter. The reason the final libraries for FFPE DNA and methyl converted DNA are shorter than the initial sheared profile is due to the nicks and damage present on these molecules being converted to strand breaks prior to SRSLY adapter ligation.

REALLY-rNONE FAQS

Q: Does ClaretBio sell a complete end to end RNA-Seq kit powered by SRSLY technology?

A: Yes! We just launched the first product in our RNA-Seq product line. The product is called REALLY-rNONE. It is an end-to-end ribodepletion RNA-Seq library prep powered by SRSLY at its core.

Q: How does REALLY-rNONE work?

A: First RNA is converted to 1st strand cDNA. After cDNA synthesis our proprietary SRSLY NGS splint adapters are ligated directly to the 1st strand cDNA. Post adapter ligation and prior to index PCR ribosomal RNA is removed via subtractive hybridization. Visit the Really Technology page for more information

Q: Why is there no second strand cDNA synthesis in REALLY-rNONE workflow?

A: Unlike traditional RNA-Seq methods that rely on T/A ligation to duplexed NGS adapters post 1st and 2nd strand cDNA synthesis, our proprietary SRSLY NGS splint adapters are ligated directly to ssDNA. Therefore, there is no need in the REALLY rNONE workflow to create and destroy a 2nd cDNA strand in order to create efficient RNA-Seq libraries and maintain directionality.

Q: What are the specifications of the REALLY-rNONE kit?

A: The REALLY-rNONE kit comes with everything needed to perform 1st strand cDNA synthesis, rRNA depletion, NGS adapter ligation, and even bead purification – yep, DNA purification beads are included in the rNONE bundle. Index PCR primers (UDI or UMI) must be purchased separately though.

Input Amount: 10-250ng
Protocol Time: ~6hrs
Input Type: RIN 1 -8

Q: Can I see the SRSLY protocol manual?

A: Unfortunately the full unabridged protocol is only available to customers post purchase. Those interested in the REALLY-rNONE protocol basics can contact customer support (technicalsupport@claretbio.com) to receive a copy of the abridged quick start protocol.

 Q: Can UMI’s be incorporated into REALLY rNONE libraries?

 A: Yes, our standard UMI add on kit is 100% integrable but also entirely optional to the REALLY-rNONE workflow.

Q: What are your recommendations for preparing RNA for REALLY-rNONE?

A: RNA extraction techniques vary widely depending on the Kingdom and Phylum of the organism. Please follow the appropriate standard protocols for total RNA extraction from your study organism.

We recommend total RNA be quantified post extraction using either the Qubit RNA HS or BR assay kit and manufacturer’s instructions. RNA integrity should be measured using a TapeStation or Bioanalyzer and associated reagents. RIN greater than 7 is preferred input. 

We have now included an optional, but recommended DNase I treatment module to remove any remaining genomic DNA prior to RNA-Seq to reduce spurious effects of DNA.

Q: Why are there three sets of beads in the REALLY-rNONE kit?

A: The REALLY workflow employs magnetic beads at various steps of the protocol:

1. Clarefy™ DNA purification beads - These magnetic beads are used for removal of proteins, enzymes and buffers after the first-strand cDNA synthesis, Ligation and index PCR steps

2. Streptavidin beads: These magnetic beads are used in the ribosomal RNA (cDNA) removal steps. These beads bind to the biotinylated rRNA probes and enable efficient pull-down

3. NEW! rNONE purification beads: These magnetic beads are used to purify the depleted cDNA pool. These are similar to the Clarefy™ DNA purification beads but now now allow purification in smaller volumes enabling better through-flex and automation.