PMID- 23731822
OWN - NLM
STAT- MEDLINE
DCOM- 20131105
LR  - 20211021
IS  - 1471-2105 (Electronic)
IS  - 1471-2105 (Linking)
VI  - 14
DP  - 2013 Jun 3
TI  - Routine performance and errors of 454 HLA exon sequencing in diagnostics.
PG  - 176
LID - 10.1186/1471-2105-14-176 [doi]
AB  - BACKGROUND: Next-generation sequencing (NGS) has changed genomics significantly. 
      More and more applications strive for sequencing with different platforms. Now, 
      in 2012, after a decade of development and evolution, NGS has been accepted for a 
      variety of research fields. Determination of sequencing errors is essential in 
      order to follow next-generation sequencing beyond research use only. This study 
      describes the overall 454 system performance of using multiple GS Junior runs 
      with an in-house established and validated diagnostic assay for human leukocyte 
      antigen (HLA) exon sequencing. Based on this data, we extracted, evaluated and 
      characterized errors and variants of 60 HLA loci per run with respect to their 
      adjacencies. RESULTS: We determined an overall error rate of 0.18% in a total of 
      118,484,408 bases. 31.3% of all reads analyzed (n=349,503) contain one or more 
      errors. The largest group are deletions that account for 50% of the errors. 
      Incorrect bases are not distributed equally along sequences and tend to be more 
      frequent at sequence ends. Certain sequence positions in the middle or at the 
      beginning of the read accumulate errors. Typically, the corresponding quality 
      score at the actual error position is lower than the adjacent scores. 
      CONCLUSIONS: Here we present the first error assessment in a human 
      next-generation sequencing diagnostics assay in an amplicon sequencing approach. 
      Improvements of sequence quality and error rate that have been made over the 
      years are evident and it is shown that both have now reached a level where 
      diagnostic applications become feasible. Our presented data are better than 
      previously published error rates and we can confirm and quantify the often 
      described relation of homopolymers and errors. Nevertheless, a certain depth of 
      coverage is needed, in particular with challenging areas of the sequencing 
      target. Furthermore, the usage of error correcting tools is not essential but 
      might contribute towards the capacity and efficiency of a sequencing run.
FAU - Niklas, Norbert
AU  - Niklas N
AD  - Red Cross Transfusion Service for Upper Austria, Krankenhausstrasse 7, 4017 Linz, 
      Austria. norbert.niklas@o.roteskreuz.at
FAU - Proll, Johannes
AU  - Proll J
FAU - Danzer, Martin
AU  - Danzer M
FAU - Stabentheiner, Stephanie
AU  - Stabentheiner S
FAU - Hofer, Katja
AU  - Hofer K
FAU - Gabriel, Christian
AU  - Gabriel C
LA  - eng
PT  - Evaluation Study
PT  - Journal Article
DEP - 20130603
PL  - England
TA  - BMC Bioinformatics
JT  - BMC bioinformatics
JID - 100965194
RN  - 0 (HLA Antigens)
SB  - IM
MH  - *Exons
MH  - HLA Antigens/*genetics
MH  - High-Throughput Nucleotide Sequencing/*methods
MH  - *Histocompatibility Testing
MH  - Humans
MH  - Sequence Analysis, DNA/*methods
PMC - PMC3679934
EDAT- 2013/06/05 06:00
MHDA- 2013/11/06 06:00
PMCR- 2013/06/03
CRDT- 2013/06/05 06:00
PHST- 2012/11/23 00:00 [received]
PHST- 2013/05/30 00:00 [accepted]
PHST- 2013/06/05 06:00 [entrez]
PHST- 2013/06/05 06:00 [pubmed]
PHST- 2013/11/06 06:00 [medline]
PHST- 2013/06/03 00:00 [pmc-release]
AID - 1471-2105-14-176 [pii]
AID - 10.1186/1471-2105-14-176 [doi]
PST - epublish
SO  - BMC Bioinformatics. 2013 Jun 3;14:176. doi: 10.1186/1471-2105-14-176.