Hello, I hope someone might be able to help me with these issues, as I'm relatively new at Bioinformatics. When analyzing data on the main website of Galaxy (all samples from the same Illumina MiSeq run) some sets fail in the BWA alignment. I have tried rerunning my workflow again, reuploading the FastQ files (in case they were corrupted) but BWA fails every single time. I've pasted the error log below. Q1: Why are most of my datasets aligned by BWA without a problem, but some consistently fail? So, some people at SEQanswers suggested I install a local Galaxy client on my computer, and I'm now trying to rework my workflow so I can use BFAST as an aligner. BWA was giving us some trouble due to indels in our mtDNA sequence, so we are trying to find another aligning tool that is more capable of working with these indels, anyway. However, now I'm running into a second problem. I have PE data, and after grooming both FASTQ files, the FASTQ Joiner generates an empty file. I did try the workaround mentioned here ( http://lists.bx.psu.edu/pipermail/galaxy-user/2012-April/004519.html) but I'm not sure I understand the following instructions "NGS: QC and manipulation -> Tabular to FASTQ" run twice Recreate both FASTQ files from the same tabular file." I ran FASTQ to Tabular with two columns, and joined the files on the first column. Why/how should I recreate both FASTQ files if I wanted to create just one single file to use as input for alignment? FASTQ Groomer doesn't seem to be able to groom the data either "Based upon quality and sequence, the input data is valid for: None Input ASCII range: '0'(48) - 'N'(78) Input decimal range: 15 - 45" Q2: How do I create a single file as input for the aligner? Thanks in advance for any help, Hilde --- The alignment failed. Error generating alignments. [bwa_sai2sam_pe_core] convert to sequence coordinate... [infer_isize] (25, 50, 75) percentile: (2402, 5234, 8910) [infer_isize] low and high boundaries: 151 and 21926 for estimating avg and std [infer_isize] inferred external isize from 251269 pairs: 5940.068 +/- 4124.607 [infer_isize] skewness: 0.511; kurtosis: -0.744; ap_prior: 1.00e-05 [infer_isize] inferred maximum insert size: 23222 (4.19 sigma) [bwa_sai2sam_pe_core] time elapses: 1.37 sec [bwa_sai2sam_pe_core] changing coordinates of 0 alignments. [bwa_sai2sam_pe_core] align unmapped mate... [bwa_paired_sw] 3297 out of 10352 Q17 singletons are mated. [bwa_paired_sw] 0 out of 188377 Q17 discordant pairs are fixed. [bwa_sai2sam_pe_core] time elapses: 2241.09 sec [bwa_sai2sam_pe_core] refine gapped alignments... 1.37 sec [bwa_sai2sam_pe_core] print alignments... 1.76 sec [bwa_sai2sam_pe_core] 262144 sequences have been processed. [bwa_sai2sam_pe_core] convert to sequence coordinate... [infer_isize] (25, 50, 75) percentile: (2595, 7186, 11297) [infer_isize] low and high boundaries: 151 and 28701 for estimating avg and std [infer_isize] inferred external isize from 84557 pairs: 7236.733 +/- 4785.357 [infer_isize] skewness: 0.066; kurtosis: -1.262; ap_prior: 1.00e-05 [infer_isize] inferred maximum insert size: 27144 (4.16 sigma) [bwa_sai2sam_pe_core] time elapses: 0.38 sec [bwa_sai2sam_pe_core] changing coordinates of 0 alignments. [bwa_sai2sam_pe_core] align unmapped mate... [bwa_paired_sw] 482 out of 3212 Q17 singletons are mated. [bwa_paired_sw] 0 out of 40389 Q17 discordant pairs are fixed. [bwa_sai2sam_pe_core] time elapses: 532.50 sec [bwa_sai2sam_pe_core] refine gapped alignments... /bin/sh: line 1: 28068 Segmentation fault bwa sampe /tmp/ 3030216.cyberstar.psu.edu/tmprNNXwj/tmpXybcjA /tmp/ 3030216.cyberstar.psu.edu/tmpq3YCcl/tmpKvAb8e /tmp/ 3030216.cyberstar.psu.edu/tmpq3YCcl/tmpvUAE3E/galaxy/main_pool/pool3/files/005/540/dataset_5540834.dat /galaxy/main_pool/pool3/files/005/540/dataset_5540837.dat >> /galaxy/main_pool/pool2/tmp/job_working_directory/004/860/4860532/galaxy_dataset_5540842.dat