BCL文件
测序产生的原始文件是BCL（binary base call）文件，测序仪在测序的时候，每个cycle都会测量编码不同颜色的通道强度，并确定最有可能的碱基类型。Real Time Analysis (RTA) 软件会将碱基类型和可信度（一个质量分数）。与FASTQ文件不同的是，BCL文件是实时产生，每个cycle的每个tile都会有一个对应文件，文件放在
<run directory>/Data/Intensities/BaseCalls/L<lane>/C<cycle>.1
文件的命名
s_<lane>_<tile>.bcl
bcl2fastq
该文件需要通过Illunima的软件或者第三方分析工具将BCL文件转成FASTQ文件。一般而言，数据下机之后，Illumina测序仪会自动将BCL转成FASTQ文件。有时候，根据实验需要，需要自己手工将BCL文件转成FASTQ文件，比如自己设计的index中含有简并碱基，或者需要调整一下转换的参数等。 Illumina提供bcl2fastq的程序包，共离线处理BCL文件，生成FASTQ文件。
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bcl2fastq -i /paht/to/run/Data/Intensities/BaseCalls/ \
-o /output/dir --sample-sheet /paht/to/run/SampleSheet.csv \
-R /paht/to/run/
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bcl2fastq文件有很多参数可调，比如在FASTQ中记录read的index(fastq文件中会记录配对的index具体序列，以及会生成额外对应的index文件)，可以添加–create-fastq-for-index-reads选项。 如果允许index有mismatch的话，可以通过–barcode-mismatches设置。 –fastq-compression-level可以设置生成的FASTQ.GZ文件的压缩比例 安装如下
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unzip bcl2fastq2-v2.17.1.14.tar.zip
tar -xvzf bcl2fastq2-v2.17.1.14.tar.gz
./bcl2fastq/src/configure --prefix=/path/to/install/
make
make install
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安装过程中，如果遇到一下问题，请更新gcc版本 问题1 cc1plus: error: unrecognized command line option “-std=c++11” 问题2 undefined reference to `boost::re_detail::perl_matcher collect2: error: ld returned 1 exit status 软件选项说明如下：
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BCL to FASTQ file converter
bcl2fastq v2.17.1.14
Copyright (c) 2007-2015 Illumina, Inc.
Command-line options:
-h [ --help ] produce help message and exit
-v [ --version ] print program version information
-l [ --min-log-level ] arg (=INFO) minimum log level
recognized values: NONE, FATAL, ERROR, WARNING, INFO, DEBUG, TRACE
-i [ --input-dir ] arg (=<runfolder-dir>/Data/Intensities/BaseCalls/)
path to input directory
-R [ --runfolder-dir ] arg (=./) path to runfolder directory
--intensities-dir arg (=<input-dir>/../) path to intensities directory
if intensities directory is specified, also input directory must be specified
-o [ --output-dir ] arg (=<input-dir>) path to demultiplexed output
--interop-dir arg (=<runfolder-dir>/InterOp/) path to demultiplexing statistics directory
--stats-dir arg (=<output-dir>/Stats/) path to human-readable demultiplexing statistics directory
--reports-dir arg (=<output-dir>/Reports/) path to reporting directory
--sample-sheet arg (=<runfolder-dir>/SampleSheet.csv)
path to the sample sheet
--aggregated-tiles arg (=AUTO) tiles aggregation flag determining structure of input files
recognized values:
AUTO Try to detect correct setting
YES Tiles are aggregated into single input file
NO There are separate input files for individual tiles
-r [ --loading-threads ] arg (=4) number of threads used for loading BCL data
-d [ --demultiplexing-threads ] arg number of threads used for demultiplexing
-p [ --processing-threads ] arg number of threads used for processing demultiplexed data
-w [ --writing-threads ] arg (=4) number of threads used for writing FASTQ data
this must not be higher than number of samples
--tiles arg Comma-separated list of regular expressions to select only a subset of the tiles
available in the flow-cell. Multiple entries allowed, each applies to the corresponding base-calls.
For example:
* to select all the tiles ending with '5' in all lanes:
--tiles [0-9][0-9][0-9]5
* to select tile 2 in lane 1 and all the tiles in the other lanes:
--tiles s_1_0002,s_[2-8]
--minimum-trimmed-read-length arg (=35) minimum read length after adapter trimming
--use-bases-mask arg Specifies how to use each cycle.
--mask-short-adapter-reads arg (=22) smallest number of remaining bases (after masking bases below the minimum
trimmed read length) below which whole read is masked
--adapter-stringency arg (=0.9) adapter stringency
--ignore-missing-bcls assume 'N'/'#' for missing calls
--ignore-missing-filter assume 'true' for missing filters
--ignore-missing-positions assume \[0,i\] for missing positions, where i is incremented starting from 0
--ignore-missing-controls assume 0 for missing controls
--write-fastq-reverse-complement Generate FASTQs containing reverse complements of actual data
--with-failed-reads include non-PF clusters
--create-fastq-for-index-reads create FASTQ files also for index reads
--find-adapters-with-sliding-window find adapters with simple sliding window algorithm
--no-bgzf-compression Turn off BGZF compression for FASTQ files
--fastq-compression-level arg (=4) Zlib compression level (1-9) used for FASTQ files
--barcode-mismatches arg (=1) number of allowed mismatches per index
multiple entries, comma delimited entries, allowed; each entry is applied to the
corresponding index;last entry applies to all remaining indices
--no-lane-splitting Do not split fastq files by lane.
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软件地址 http://support.illumina.com.cn/downloads/bcl2fastq-conversion-software-v217.html
文档手册 http://support.illumina.com/content/dam/illumina-support/documents/documentation/software_documentation/bcl2fastq/bcl2fastq_letterbooklet_15038058brpmi.pdf
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