Bisulfite analysis with Illumina FastQ from different lanes

[antgomo]

Hi everybody,

I'm a newbie in NGS bisulfite Methylation analysis, so first, apologies iof the question is so simple but I couldn't find any topic related to: Anyway, we receive samples from BGI who work with Illumina, the problem working with Illumina as you may know, is the FASTQ paired-end files that we get are splitted in several lanes , I mean

NB_Lib1_L1_PE250_1.fq
NB_Lib1_L1_PE250_2.fq
NB_Lib1_L2_PE250_1.fq
NB_Lib1_L2_PE250_2.fq
NB_Lib1_L3_PE250_1.fq
NB_Lib1_L3_PE250_2.fq

Instead of NB_Lib1_PE250_1.fq and NB_Lib1_PE250_2.fq, I followed the indications from other threads at Seqanswers and Biostar and I concatenated these 6 files in two ( 3 for the first paired-end and 3 for the second), the problem is that I got fastq files of 71 Gb, and I don't know if it is the proper way to do bisulfite analysis using BISMARK or Methylcoder, as far I know, Illumina has a pipeline with Bismark for bisulfite analysis, but following this it is mandatory to use CASAVA and I don't want to. So, my questions are:

Do I have to work using this procedure, concatenating the files in spite of their weight, or is there any other procedure of working with this splitted Illumina fastq (i.e, analyse FASTQ from one lane, then another lane and so on, the problem is, how to concatenate the final results of this)

Thanks

[fkrueger]

Hi antgomo,

As far as Bismark is concerned you could either concatenate the files before the alignments (in the same way you did) or merge the result files, either will be fine. You just need to make sure that the sequences are in the correct order (every single sequence in file_1 must correspond to the other end in file_2) throughout.

so it could look like this:
-1 NB_Lib1_L1_PE250_1.fq,NB_Lib1_L2_PE250_1.fq,NB_Lib1_L3_PE250_1.fq -2 NB_Lib1_L1_PE250_2.fq,NB_Lib1_L2_PE250_2.fq,NB_Lib1_L3_PE250_2.fq

or
-1 NB_1.fq -2 NB_2.fg (for concatenated files)


Just for the record, as far as I know the pipeline recommended by Illumina uses CASAVA only for their RRBS (do you have RRBS-type libraries?). If you need any additional help with your files (e.g. regarding quality control etc...) please send me an email.