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36 CHAPTER 2. A HIGH-THROUGHPUT DNA SEQUENCING DATA ANALYSIS SUITE 1 #?sample read barcode Col-0 2 TTCACG Col-0 2 GGATGT Ler-1 2 CTAGGC 5 Ler-1 2 AGACCA Listing 2.1: Example of a Simple Demultiplexing Sheet 2.4.2 A Format for Description of Multiplexing Setups A SHORE demultiplexing sample sheet is a simple tab-delimited text table with named columns. The recognized column names are lane, sample, barcode_group, read, barcode, extbarcode and barcode_type. The sample sheet is parsed utilizing a generic table input API provided by the SHORE library. Column names are dened in the header line, the rst non-empty line of the le that is not a line comment, or is introduced with a special comment tag #?. Columns are recognized by name and may occur in arbitrary order; further columns may be present, but are ignored. The only mandatory column, named sample, denes the sample identiers that bar codes are to be translated to. All further columns may be added in arbitrary combinations and order. A typical simple index read demultiplexing conguration is described by additionally specifying the read and barcode columns (listing 2.1), with read specifying the index of the read that contains the bar code tag and barcode the actual sequence of the bar code oligomer. The read index column may be omitted, indicating that all sequence reads of a pair are attached to identical bar codes. Index read and 5 ′ bar coding require suitable treatment of the respective bar code oligomers. While 5 ′ bar codes must be clipped, with the remaining part of the read sequence to be retained, index reads must be removed completely from the data set. The default implemented in SHORE is to apply bar code clipping if the sample sheet entry refers to the rst read or if the read index was omitted, and to lter bar-coded reads where the read index is greater than one. The barcode_type column allows to explicitly control this behavior. SHORE accepts bar code types read, 5prime and none, where the type is associated with the read index, i. e. all of a lane's entries with the same read index must also specify the same bar code type. Bar codes of type read will trigger ltering of the entire bar code associated read, whereas 5prime bar codes will be clipped. For congurations with bar code information distributed across multiple sequence reads, several entries with dierent read index values are for each sample added to the denition (listing 2.2, lines 47). The sample sheet entries will then be grouped on the sample identier, with each possible combination of bar codes with diering read indexes considered a valid bar code tuple for the respective sample. Sample sheet entries with the special sample identier * are interpreted as valid for each sample specied for the respective sequencing lane (e. g. lines 1617). For example, listing 2.2 denes two valid bar codes for the Ler-1 sample for each of the three sequencing reads, and thus 2 3 = 8 valid 3-tuples of bar codes that can be resolved to that sample. Automatic combination of all entries listed for a sample can be controlled by the user via specication of the barcode_group column. Sample sheet entries with dierent bar code group values are not able to form a valid bar code tuple (e. g. listing 2.2, lines 1013). For example, the rst read bar code specied in line 10 for the Col-0 sample may only combined with the second read entry from line 11 and not the one from line 13. Furthermore, a combination of line 10 and 13 would collide with the bar code tuples including entries from line 4 and 7, which are already specied to resolve to the Ler-1 sample. As with the sample column, the bar code group may be set to * to specify and entry that refers to all groups of the respective sample in the respective
2.4. A FLEXIBLE SEQUENCING READ DEMULTIPLEXING SYSTEM 37 1 #?lane sample barcode_group read barcode extbarcode barcode_type # Bar codes for the Ler-1 sample. 1 Ler-1 0 1 AACT TGCAG 5prime 5 1 Ler-1 0 1 TAGC TGCAG 5prime 1 Ler-1 0 2 AACT * read 1 Ler-1 0 2 CCCT * read # Bar codes for the Col-0 sample. 10 1 Col-0 0 1 AACTT GCAG 5prime 1 Col-0 0 2 GGAC * read 1 Col-0 1 1 TTGCT GCAG 5prime 1 Col-0 1 2 CCCT * read 15 # Third read has the same bar codes for all valid samples. 1 * * 3 GGAC * 5prime 1 * * 3 TTGC * 5prime # Lane 2 is not multiplexed, discard the index read. 20 2 Bur-0 0 2 * * read Listing 2.2: Example of a Full Demultiplexing Sheet lane (e. g. lines 1617). For certain applications, the identity of several nucleotides immediately following 5 ′ bar code sequences is known, like for example restriction site sequence in RAD-Seq (section 1.3). While such sequences can be exploited to correctly assign each read to the appropriate sample, they usually should in contrast to the bar code oligomers not be removed from the output. Parts of the recognition sequence that should not be clipped from the read can be specied in the extended bar code (extbarcode) eld of the table. Internally, the sequence to be recognized is contructed by concatenation of the barcode and extbarcode elds, while the division of sequence among both elds is translated into a bar code cut position. For bar code types other than 5prime, the split into bar code and extended bar code has no eect. The bar code cut position is determined in the context of the respective bar code tuple, i. e. for the same recognition sequence dierent samples may dene a dierent split between bar code and extended bar code, as demonstrated by lines 4 and 10 of listing 2.2. If no part of the recognition sequence is to be removed from the output, then the entire oligomer can be provided as extended bar code, with column barcode either omitted or set to * . If neither bar code nor extended bar code are provided with a value other than * , then the respective sample sheet entry will match any read sequence. With bar code type either none or 5prime, such an entry can be utilized for assigning a certain sample identier to an entire sequencing lane. On the other hand, this property may be exploited to completely remove all reads with a certain read index from the output (e. g. listing 2.2, line 20). The sample sheet column lane serves to allow independent demultiplexing specications for dierent sequencing lanes in a single sample sheet le. Rows with diering sequencing lane elds are completely independent of each other. If the sequencing lane column is omitted, then the entire demultiplexing specication is considered valid for all lanes of the instrument run.
Page 1: An Integrated Data Analysis Suite a
Page 4 and 5: Erklärung Hiermit erkläre ich, da
Page 7 and 8: Acknowledgment First of all I would
Page 9: Abstract The various parallel DNA s
Page 12: xii CONTENTS 2.6 A Parallelization
Page 16 and 17: 2 CHAPTER 1. INTRODUCTION 1.2 Appro
Page 18 and 19: 4 CHAPTER 1. INTRODUCTION genetic a
Page 20 and 21: 6 CHAPTER 1. INTRODUCTION Instrumen
Page 22 and 23: 8 CHAPTER 1. INTRODUCTION biases. S
Page 24 and 25: 10 CHAPTER 1. INTRODUCTION To achie
Page 26 and 27: 12 CHAPTER 1. INTRODUCTION quantiti
Page 28 and 29: 14 CHAPTER 1. INTRODUCTION of featu
Page 30: 16 CHAPTER 1. INTRODUCTION Our appl
Page 34 and 35: 20 CHAPTER 2. A HIGH-THROUGHPUT DNA
Page 72: 58 CHAPTER 2. A HIGH-THROUGHPUT DNA
Page 76 and 77: 62 CHAPTER 3. A C++ FRAMEWORK FOR H
Page 91 and 92: 4 Closing Remarks Routine analysis
Page 93: 79 preparation should further induc
Page 96 and 97: 82 BIBLIOGRAPHY [9] S. Brenner, M.
Page 98 and 99: 84 BIBLIOGRAPHY [29] N. Cloonan, A.
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86 BIBLIOGRAPHY [48] B. A. Methe, K
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88 BIBLIOGRAPHY [72] S. Andrews. Fa
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90 BIBLIOGRAPHY [98] M. A. DePristo
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92 BIBLIOGRAPHY [124] W. J. Kent, C
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