- A ligation-based single-stranded library preparation method to analyze cell-free DNA and synthetic oligos. - polymerase will A-tail the 3-prime ends of template DNA to promote efficient ligation of the sequencer-specific adapters [2, 3]. - 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - Full list of author information is available at the end of the article. - Single-stranded DNA library preparation methods offer several advantages over traditional dsDNA methods [5–7].. - By denaturing the duplexed template DNA prior to adapter ligation and maintaining the DNA as single strands through at least an initial adapter ligation, single-stranded preparation methods are theoretically able to convert all of the molecules captured by traditional dsDNA library preparation methods as well as nicked dsDNA and ssDNA molecules. - Further, the sequencing reads from some ssDNA library methods represent the natural 5- prime and 3-prime ends of the input DNA fragments. - Thus, when mapped to a reference genome, these data reveal the exact genomic location of the input fragments. - Several single-stranded library preparation methods have been described since . - Also, in some cases special bioinformatic processing of the data is required to deal with artifacts introduced as a conse- quence of library prep [22, 28].. - thermostable single-stranded binding protein (SSB).. - Both sequencing adapters are dsDNA, except for a single-stranded splint overhang of random nucleotides that occurs on the 3- prime termini of the bottom strand of forward adapter and the 5-prime termini of the bottom strand of the re- verse adapter. - Simultaneously, the random nucleotides of the splint. - adapter anneal to the single-stranded template molecule.. - Performance of the SRSLY protocol. - The data generated resulted in about 15-fold coverage of the human genome for both SRSLY and NEBNext Ultra II samples per cfDNA extract and about 5-fold coverage for both TaKaRa SMARTer and Swift 1S per cfDNA extract.. - 2a and its inset, the two preparation methods differ in the proportion of reads captured at different fragment lengths, as well as the length distribution of the sub-peaks present in the sawtooth pattern. - Figure 2b shows that SRSLY produces fold-coverage similar to that of the NEBNext Ultra II kit and that both methods produce relatively uniform gen- omic coverage. - Figure 2d shows that the GC content of SRSLY libraries is similar to that of the NEBNext Ultra II kit. - However, the 5-prime overhang “fill-in” and the 3-prime overhang exonuclease activity of T4 DNA polymerase generates a 3-prime end that is not representative of the original molecule when overhangs of either type are present. - In this way, the end- polishing procedure is expected to make all 3-prime ends mirror what is present at the 5-prime end of the comple- mentary strand.. - First, for both SRSLY and NEBNext there is significant deviation from the average base composition at the start of each read, as well as upstream of the biological fragmentation point. - This is a well-documented feature of the cfDNA nucleosome protection model fur- ther discussed in the cfDNA results section below. - Sec- ond, unlike the dsDNA library data, the average base composition for the start of the forward reads and the start of reverse reads differ in SRSLY libraries. - Third, the average base composition for the start of the forward read in NEBNext Ultra II libraries are exactly the reverse-complement of the average base com- position for the start of the reverse read. - Finally, the average base composition for the start of the forward read in SRSLY libraries is nearly identical to that of NEBNext Ultra II libraries. - We compared the length distribution, read coverage, com- plexity, GC content, and DNA termini results of the SRSLY prep to those of the TaKaRa SMARTer and Swift 1S methods as well. - 3b) confirm that SRSLY produces reduced coverage across the overhanging regions compared to the double- stranded regions of the synthetic oligos illustrating the method’s ability to yield stranded data that accurately characterizes the input DNA. - Three-prime exonuclease activity, on the other hand, causes nearly complete loss of the 3-prime overhang se- quence when it is present.. - Single-stranded oligo libraries. - Analysis of the propor- tion of template lengths from sequencing these libraries shows that the SRSLY protocol generates ssDNA librar- ies across this length range (Fig. - These truncated DNA fragments have lengths that are nearly uniformly distributed across the length of the oligo. - 3 Coverage of duplexed oligos containing single-stranded overhangs for SRSLY and NEBNext. - We hypothesize that these two observations demonstrate the limits of the phosphoroamadite method of oligo synthesis. - 4 Single-stranded oligo analyses by the SRSLY method. - From these data, we explored the ability of SRSLY libraries to reveal aspects of the positioning of nucleosomes and other DNA-binding proteins.. - observed this oscillation pattern in our SRSLY data we ex- amined the A/T and G/C genomic dinucleotide in mole- cules of three fragment lengths and 83 bp, including bases 100 nts upstream and downstream of each of the three read lengths (Fig. - We centered each on the midpoint of the sequence. - We also observe a strong oscillation signal for ~ 55 bp upstream of the 83 bp fragment length indicating that these molecules are likely derived from degraded nucleosomal associated. - Negative numbers denote genomic regions upstream (5-prime) of the midpoint and positive numbers denote genomic regions downstream (3-prime) of the midpoint. - Due to the dsDNA end-polishing step, the terminal pro- file of the 5-prime and 3-prime ends in NEBNext data are mirror images of each other (Fig. - Compar- ing our WPS results with previously published results using an alternative ssDNA library protocol, we observe good con- cordance with respect to the location of the peaks and troughs (Fig. - While SRSLY generates sequen- cing library molecules from single-stranded DNA fragments, it can be used for DNA that is either single- stranded, double-stranded or a combination of the two.. - We present validation of the SRSLY method via com- parison to traditional dsDNA library preparation methods and a commercially available ssDNA preparation method showing that SRSLY produces sequencing libraries with uniform coverage, higher complexity, and base compos- ition similar to those of the widely used NEBNext Ultra II kit. - By cal- culating the depth of coverage at each position for synthetic duplex oligos containing single-stranded DNA overhangs we showed that SRSLY is able to retain strand information from dsDNA and a more accurate characterization of the template molecules. - Further analysis of data generated from SRSLY may reveal further details of the nature of the over- hangs present in cfDNA molecules and perhaps the iden- tities of the active nucleases that generate them.. - Concentration of the purified cell- free DNA (cfDNA) was measured using the Quant-iT high sensitivity dsDNA Assay Kit and a Qubit Fluorometer (ThermoFisher Scientific). - The forward SRSLY adapter contains a 5-prime overhang in the splint portion of the adapter and a free 3- prime OH end on the ligating end. - The re- verse SRSLY adapter contains a 3-prime overhang in the splint portion of the adapter and is 5-prime phosphory- lated for ligation. - Working stocks of the adapters are made by diluting the adapters in TE + 50 mM NaCl.. - On ice, 1 pmol of the forward and 1 pmol of the reverse SRSLY adapters were added to the denaturation reaction, as well as PEG-8000, T4 DNA ligase Buffer, T4 PNK, and T4 DNA ligase (all New England Biolabs) to a final vol- ume of 50 μl. - The base composition per position was normalized with the mode for that base along the length of the region and log-2 transformed. - Double-stranded synthetic oligo sequencing coverage at each position in the oligo was determined utilizing a custom script akin to samtools depth and plotted in R utilizing ggplot2 as a function of percent across the length of the oligos in 0 base coordinates.. - CG/GC/GG) dinucleotide interaction such that the cen- ter of the insert was at 0 and the regions upstream of. - Synthetic single-stranded oligos sequences.. - Synthetic single-stranded oligo raw read counts. - SRSLY: Single Reaction Single-stranded Library. - SSB: Single-stranded binding protein. - ssDNA: Single- stranded DNA. - https://doi.org/10.. - https://doi.org/10.1186/gb r119.. - 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