The quality and quantity of library were checked by Agilent 2100 Bioanalyzer and Roche LightCycler ® 480 II Real-Time PCR system according to the manufacturer's instructions. PCR amplified fragments were digested with MmeI, end-repaired, A-tailed and ligated with P1 and P2 sequencing adaptors to make the palindromic library for sequencing by SOLiD 5500xl.
#Palindromic sequence plus
The inserted DNA in yT&A vector was amplified with PCR using M13 forward (5'-GTTTT CCCAG TCACG AC-3') and reverse (5'-TCACA CAGGA AACAG CTATG AC-3') primers and EconoTaq™ PLUS GREEN 2X Master Mix (Lucigen Corp.). coli genome: Tag1 (18 bp, 5'-GGCAA TGGCA CCATC GCT-3') and Tag2 (17 bp, 5'-CCACG ACCGC TGAGG TT-3', or 5'-AACCT CAGCG GTCGT GG-3' on the complementary strand which was sequenced by SOLiD 5500xl sequencer), making up a total length of 61 bp. This clone contains a centralized palindromic sequence (26 bp, 5'-AGTCG GAGTC TGCGC AGACT CCGAC T-3') flanked by two tags originated from the E. Co., Ltd.) and verified by traditional Sanger sequencing using ABI PRISM ® 96-capillary 3730xl DNA Analyzer (Notice that this step proved the capability of a traditional sequencing-by-synthesis method to sequence through the palindromic region). Paired-end ditag DNA fragment of 5'-GGCAA TGGCA CCATC GCT AG TCGGA GTCTG CGCAG ACTCC GACTC CACGA CCGCT GAGGT T-3' was cloned into yT&A vector (Cat. Here we report the results, indicating the incapability for a sequencing-by-ligation machine to read through the palindromic region.Ĭlone preparation and sequencing library construction for the palindromic library The library was sequenced by SOLiD 5500xl sequencer. We constructed a single clone containing a palindromic region flanked by two tag sequences (~18 bp), amplified the clone, digested with MmeI, and constructed a sequencing library from the MmeI fragments. In this article, we used centralized palindromic sequence to study the effects of a palindromic sequence on sequencing. Thus, it is an important issue to further understand the potential obstacles caused by palindromic sequences, especially for the sequencing-by-ligation mechanism. mRNAs or miRNAs) may become regional obstacles for sequencing-by-ligation sequencers, causing problems in genome assembly and introducing bias in transcriptome analysis. Given the fact that short stretches of palindromic sequences may form hairpin structures in the single-stranded template, palindromic sequences which naturally exist in genomes or in RNA sequences (esp. The success of ligation has to rely on the availability of its complementary strand. It has a potential to form a hairpin structure. A palindromic sequence is a nucleic acid sequence that reads the same no matter from the 5' end of the sequence itself or from the 5' end of its complementary strand. However, the potential obstacles imposed by palindromic regions have never been addressed.
Potential underrepresentation of GC-rich regions have been reviewed for both sequencing-by-synthesis and sequencing-by-ligation mechanisms. Since DNA polymerase is an essential enzyme in the cell, the former is considered as a more natural approach compared with the latter. The most evident difference between sequencing-by-synthesis and sequencing-by-ligation is that the former uses DNA polymerase to incorporate complementary nucleotides to the elongating strand, while the latter uses ligase to seal the junction between the elongating strand and the newly incorporated complementary oligonucleotides. This sequencing approach, which grows in 3' to 5' direction, is coined as "sequencing-by-ligation" mechanism. On the other hand, although SOLiD systems also use emulsion PCR to amplify templates on beads, its sequencing is extended by two unique steps: first by using oligos to hybridize to the target in the single-stranded template, followed by using ligase to seal the nick between the 5' of the growing strand and the 3' of the oligo. As such, both 454 and Solexa lineages adopt "sequencing-by-synthesis" approach. Solexa lineage adopts bridge PCR to amplify temples on slide followed by colorimetric sequencing with DNA polymerase. 454 uses emulsion PCR to amplify temples on beads followed by pyrosequencing with DNA polymerase. The sequencing chemistries of these three systems more or less differ. In general, the cost for 454 is much higher than the other two, but its sequence length can easily reach 400 bps, followed by the Solexa lineage and then SOLiD, which can only produce short reads up to 75 bp. Currently, the sequencing market is dominated by 3 NGS lineages: Roche's 454, Illumina's Solexa, and Life Technologies' SOLiD. Next-Generation Sequencing (NGS) promises to provide robust sequencing platforms for basic biological researches as well as clinical and molecular diagnoses.
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